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UNIT 12.12From In Vitro to In Vivo: Imaging fromthe Single Cell
to the Whole OrganismJung Julie Kang,1 Ildiko Toma,1 Arnold Sipos,1
and Janos Peti-Peterdi11University of Southern California, Los
Angeles, California
ABSTRACTThis unit addresses the applications of fluorescence
microscopy and quantitative imag-ing to study multiple
physiological variables of living tissue. Protocols are
presentedfor fluorescence-based investigations ranging from in
vitro cell and tissue approaches toin vivo imaging of intact
organs. These include the measurement of cytosolic parame-ters both
in vitro and in vivo (such as calcium, pH, and nitric oxide),
dynamic cellularprocesses (renin granule exocytosis), FRET-based
real-time assays of enzymatic activity(renin), physiological
processes (vascular contraction, membrane depolarization), andwhole
organ functional parameters (blood flow, glomerular filtration).
Multi-photon mi-croscopy is ideal for minimally invasive and
undisruptive deep optical sectioning of theliving tissue, which
translates into ultra-sensitive real-time measurement of these
param-eters with high spatial and temporal resolution. With the
combination of cell and tissuecultures, microperfusion techniques,
and whole organ or animal models, fluorescenceimaging provides
unmatched versatility for biological and medical studies of the
livingorganism. Curr. Protoc. Cytom. 44:12.12.1-12.12.26. C 2008 by
John Wiley & Sons,Inc.
Keywords: in vivo imaging in vitro imaging multiphoton
fluorescencemicroscopy real-time imaging intravital imaging
laser-scanning microscopy
INTRODUCTIONThe application of in vitro fluorescence imaging to
cellular studies permits importantdiscoveries about structure,
function, responses to the environment, intracellular signal-ing
pathways, and, indirectly, intercellular relationships. Such
studies are particularlyuseful in determining the specific
mechanisms by which particular cellular componentscontribute to
larger processes, such as the roles of endothelial nitric oxide
productionin vasodilation or the relevance of vascular smooth
muscle calcium concentration tovasoconstriction. Furthermore, both
the acute and chronic effects of various stimuli maybe
investigated: varying the culturing media or conditions of cells
may cause changes incell signaling or function, which can be
detected with quantitative imaging. Fluorescenceimaging has
tremendous applicability to studies of cytosolic changes (e.g.,
calcium, pH,cell volume), cellular responses to stimuli (e.g.,
nitric oxide, prostaglandins), and proteincontent/enzyme activity
(e.g., renin). Cellular studies permit the determination of
themechanisms responsible for propagating complex pathways,
providing valuable targetsfor intervention, especially in
disease.
This unit delineates several different protocols for applying
fluorescence imaging technol-ogy to cellular studies. The superior
image quality and resolution permits the visualizationof morphology
in living cells, as shown with primary cell cultures of vascular
smoothmuscle in Figure 12.12.1 A,B. The acidophilic fluorophore,
quinacrine, may be usedto label renin granules in appropriate
cells, shown in Figure 12.12.1 C. Lipid vesiclesmay be identified
with the red stain, Nile Red, shown in Figure 12.12.1D. The
judiciousselection of dyes permits simultaneous co-labeling of
multiple intracellular structures,
Current Protocols in Cytometry 12.12.1-12.12.26, April
2008Published online April 2008 in Wiley Interscience
(www.interscience.wiley.com).DOI:
10.1002/0471142956.cy1212s44Copyright C 2008 John Wiley & Sons,
Inc.
Cellular andMolecularImaging
12.12.1Supplement 44
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12.12.2Supplement 44 Current Protocols in Cytometry
Figure 12.12.1 Fluorescence imaging of primary cultures and cell
lines to study morphologyand compartments. DIC (A) and uorescence
(B) images of primary vascular smooth musclecells derived from
manually dissected arteriolar explants. (C) Quinacrine-stained
renin granules inAs 4.1 cells, a renin-secreting tumor cell line.
(D) Renal medullary interstitial cells in culture labeledwith
Nile-Red staining of lipid vesicles. (E) A mouse macula
densa-derived cell line is co-labeledwith Mito-Tracker Red
(mitochondria) and quinacrine (green, acidic granules). For a color
versionof this gure, see http://www.currentprotocols.com.
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12.12.3Current Protocols in Cytometry Supplement 44
Table 12.12.1 Dyes Used in Cellular Cuvette-Based
SpectrouorometryStudies
Dye Parameter measured Excitation Emission
Renin-FRETSubstrate
Renin enzymatic activity 340 nm 490 nm
Fura-2 Intracellular calcium( ratio = Ca2+)
340/380 nm 510 nm
Fluo 4 Intracellular calcium 488 nm 516 nmDAF-FM Nitric oxide
495 nm 515 nmBCECF pH ( ratio = pH) 500/440 nm 530 nm
and the use of Mito-Tracker Red for mitochondria and quinacrine
for acidic granules inmacula densa cells beautifully illustrates
the morphology and highlights the specificityof each of these dyes
for their organelles (Fig. 12.12.1E). The imaging system may alsobe
used to visualize fixed cells for immunocytochemistry experiments.
In addition tostructural characterization, the appropriate
selection of fluorescent probes permits theapplication of
cuvette-based spectrofluorometry to investigate direct
cause-and-effectrelationships by studying cytosolic signals and
second messengers in response to dif-ferent stimuli. Table 12.12.1
lists various cellular structural and messenger dyes usefulfor
spectrofluorometry studies. Quantification of cellular enzymatic
activity is anothervaluable application of this technique. A
recently developed fluorogenic peptide based onfluorescence
resonance energy transfer (FRET) permits real-time measurements of
reninactivity and can be used to analyze enzymatic activity of
tissue samples from healthy anddiseased animals. Imaging studies on
cells have limitless applicability to studying directand acute
effects as well as evaluating changes in chronic disease
conditions.
STRATEGIC PLANNINGCuvette-Based Spectrouorometry to Assess
Second-Messenger Signalingin Living CellsSignal transduction refers
to the process by which a cell receives input and convertsit to
another signal, typically involving an ordered sequence of
intracellular reactionscarried out by enzymes and linked through
second messengers. The association of stim-ulus with signal
transduction pathway, second messenger, and ultimately,
end-response,provides important mechanistic information that can be
utilized to promote favorableand inhibit detrimental processes.
Stimuli that initiate a cellular response may be molec-ular
(hormones, cytokines), environmental (extracellular matrix), or
physical (light) innature. Some cellular responses to extracellular
stimulation that depend on signal trans-duction include metabolism
and cell proliferation/death. Therefore, the translation ofexternal
cues into internal messages that elicit specific cellular actions
involves signaltransduction. Many diverse disease processes
including diabetes, hypertension, autoim-munity, and cancer arise
from defects in signal transduction pathways, elucidating
theimportance of signal transduction to physiology as well as
pathology.
In vitro cell signalingFluorescence microscopy has vast
applicability to cell signaling studies. Alterations inpH
(Peti-Peterdi et al., 2000), sodium (Peti-Peterdi et al., 2002a),
and nitric oxide (Kovacset al., 2003) have all been investigated
with commercially available probes. Fluorescenceimaging can also be
used to study intracellular changes in response to different
stimuli.Signals such as hormones and growth factors are received at
cell surface receptors andthen relayed to target molecules in the
cytosol and/or nucleus by second messenger
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12.12.4Supplement 44 Current Protocols in Cytometry
molecules. In addition to functioning as molecules that
translate extracellular cues intointracellular messages, second
messengers greatly increase the amplitude of the signal.Major
classes of second messengers include cyclic nucleotides (e. g.,
cAMP and cGMP),inositol trisphosphate (IP3), diacylglycerol (DAG),
and calcium ions. Fluorescent dyesfor many second messengers exist,
so prudent experimental design can provide definitiveconfirmation
of the effects of hypothesized inputs on cellular behavior. For
example,the technology may be applied to investigating the effects
of a proposed substrate ona G proteincoupled receptormediated
intracellular calcium signal. Furthermore, theeffects of receptor
inhibition on the second messenger signal may be analyzed with
thisapproach. Ultimately, the data obtained can elucidate the
influence of a stimulus on acell by assessing changes in calcium
signaling in larger physiological or pathologicalprocesses
(Peti-Peterdi et al., 2002a).
Calcium signalingSince intracellular signal transduction is
largely carried out by second messengermolecules, identification of
changes in second messengers provides evidence of a di-rect effect
of stimulus on cell behavior. Calcium is one of the most widely
studied secondmessengers because it is used in a multitude of
processes, including muscle contraction,the release of
neurotransmitters, cell proliferation, secretion, cytoskeletal
management,cell movement, gene expression, and metabolism.
Intracellular calcium concentrationis normally maintained at very
low levels by sequestration in the smooth endoplasmicreticulum and
mitochondria. Three main signals that promote the activation and
releaseof calcium are G proteincoupled receptor-regulated pathways,
receptor tyrosine kinasepathways, and ligand or current-gated ion
channels. Its release from the endoplasmicreticulum results in its
binding to and activation of proteins or enzymes.
The first dye to be highly used for calcium imaging was Fura-2,
a ratiometric fluorescentdye that binds to free intracellular
calcium. Its fluorescence is detected at 510 nm inresponse to
alternate excitation at 340 and 380 nm, with the ratio of the two
(340/380) di-rectly correlating to the amount of intracellular
calcium. The use of a ratio resolves someexperimental challenges
such as dye concentration and background autofluorescence,making it
an ideal choice for quantitative measurements. Fura-2 is thus the
preferen-tial dye for ratiometric calcium imaging when the
alternation of excitation wavelengthsis more practical than the
detection of multiple emission wavelengths. A newer gen-eration of
calcium fluorophores includes fluo-4, which provides an efficient,
validatedmethod of imaging intracellular calcium fluxes. Because
fluo-4 AM loads faster andoffers greater fluorescence at equivalent
concentrations, it is the preferred indicator forconfocal
microscopy, flow cytometry, and microplate screening applications.
Althougheach fluorescent calcium probe has different benefits,
their combined use may providean extra level of validation of the
data obtained. Fluo-4 and Fura Red respond to [Ca2+]ichanges with
no significant kinetic differences. However, fluo-4 fluorescence
increaseswith a rise in [Ca2+]i, while Fura Red fluorescence
decreases. These features make Fluo-4and Fura Red an excellent dye
pair for ratiometric [Ca2+]i imaging (Peti-Peterdi, 2006).The
application and choice of various fluorophores depends on the
fluorescence equip-ment available and on the biological processes
(range of calcium changes) to be examined.Ratiometric approaches
(the simultaneous use of two different dyes or dye forms thathave
similar loading characteristics) are almost always recommended,
which excludethe possibility of artifacts associated with dye
loading, photobleaching, leakage, cellvolume changes, etc. For the
widely used xenon light source-based systems, fura-2 isan ideal
choice since excitation ratiometric approaches can be used. For
instrumentspowered by laser sources, such as confocal and
multi-photon fluorescence microscopes,emission ratiometric
approaches are typically required because of the single, fixed
ex-citation wavelength. For these systems, the fluo-4/Fura Red
ratio pair is the preferred
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12.12.5Current Protocols in Cytometry Supplement 44
selection for calcium imaging. Fluo-4 and Fura Red AM forms
(cell membrane perme-able for loading) are non-fluorescent, as
opposed to fura-2 and indo-1, and do not tendto compartmentalize,
factors that would significantly limit the sensitivity of
detectingcytosolic-free calcium. It is recommended to check the
calcium dissociation constant(Kd) of different calcium probes,
which will give an idea about the range of calcium thatthe given
fluorophore can detect. For example fura-2 (Kd = 224 nM) and fluo-4
(Kd =345 nM) are used to detect low or medium cytosolic calcium
levels while fluo-5 F is thechoice for high calcium conditions (Kd
= 2.3 M).
A Novel Application of FRET: Cuvette-Based Spectrouorometry to
EvaluateCellular Enzyme ActivityCuvette-based spectrofluorometry is
valuable for investigations that aim to determinethe presence or
absence of a direct, acute effect of a given intervention on
cellularfunction. In certain circumstances, the magnitude of the
effect may even be quantified.Cuvette-based investigations are
readily applicable for the study of specific causes andeffects in
isolated cells, providing definitive information about external
influences or thecellular machinery involved. For example, the
cuvette system has been applied to studythe potential value of
purinergic receptormediated calcium signaling as a potentialrescue
for epithelial cells in cystic fibrosis (Zsembery et al., 2003).
Alternatively, theexperimental approach has been used to assess
intact cellular machinery by comparingchanges in second messenger
signals like calcium between different receptors (Hwanget al.,
2003). Some investigative questions require more than quantitative
data, but also thevisual information from imaging. Innovative
studies have harnessed spectrofluorometrywith microscopy to
investigate cellular signaling with its environment and
epithelialcell polarity intact, such as apical and basolateral
channels, which contribute to maculadensa calcium signaling
(Peti-Peterdi and Bell, 1999). Furthermore, the approach hasbeen
applied to characterization of the behavior of cells themselves:
quantification ofenzymatic activity has tremendous value in
estimating protein content in disease models(Kang et al., 2006b,
ADDR). In contrast, imaging-based applications provide the
addedbenefits of studying the contribution of cellular polarity and
possible interactions of cellswith their environment (Peti-Peterdi
et al., 2003). It provides an excellent tool for study ofthe
mechanisms, regulation, and functional significance of
physiological phenomena likerenin release (Toma et al., 2006).
Furthermore, intercellular interactions can be
observed.Fluorescence resonance energy transfer (FRET) is a tool
based on the energy transferbetween a donor and acceptor pair of
fluorophores, which can be used to quantifymolecular dynamics like
the interactions between proteins. When the donor and
acceptorfluorophores are in close proximity to each other,
excitation of the donor results indetectable emission only from the
acceptor. The donor-acceptor pair is carefully selectedso that
donor emission falls into the specific wavelength for acceptor
excitation. Afluorescent donor is excited and its emission energy
is quenched through absorption bythe acceptor. Intermolecular FRET
from donor to acceptor results only in the detectionof emission
from the acceptor. When the donor-acceptor pair is dissociated,
FRET canno longer occur (excitation of the donor is no longer
quenched by the acceptor), anddonor emission may be detected.
Therefore, the efficiency of FRET is determined bythree parameters:
distance between the donor and acceptor, overlap between the
donoremission spectrum and acceptor absorption spectrum, and the
relative orientation of thedonor emission dipole moment to the
acceptor absorption dipole moment. FRET canbe quantified by
cuvette-based spectrofluorometry experiments or in microscopy
imageson a pixel-by-pixel basis. Essentially, the technique
capitalizes on the proximity of thefluorescent molecules and can be
applied to study protein interactions, conformationalchanges, or
enzymatic activity.
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A variation on FRET: Renin enzymatic activity
assessmentsFluorescence imaging has tremendous potential for the
qualitative and quantitative char-acterization of
pathophysiological conditions. Although typically used to study
proteinstructural interactions, the principles of FRET can be
applied to a plethora of other in-vestigations. A recently
developed fluorogenic renin substrate (Invitrogen and AnaSpec)makes
use of FRET between a donor-acceptor pair linked by a sequence of
human an-giotensinogen containing the renin cleavage site at the
Leu-Val bond (Wang et al., 1993).In imaging-based applications, the
method can be used to visualize the intact
intra-renalrenin-angiotensin system, studying the directionality of
renin release and activity (Peti-Peterdi et al., 2004). In the
absence of active renin enzyme, EDANS fluorescence isquenched by
the acceptor molecule DABCYL due to their close proximity and the
FRETbetween them. However, when cleaved by renin, the fluorophores
dissociate and give riseto bright green EDANS fluorescence. The
typical spectrofluorometry reading for a reninactivity assay is
shown in Fig. 12.12.2B. This technique allows real-time
measurementof renin activity, circumvents the use radioactivity,
and is conveniently performed withinminutes, as opposed to
conventional renin assays using radioimmuno-methods, whichrequires
several days to develop. This novel fluorogenic renin substrate has
tremendouspotential to measure renin enzymatic activity in renal
cortical tissue homogenates oreven to directly visualize the
activity of the intra-renal renin-angiotensin system. BasicProtocol
3 provides quantitative data on the enzymatic activity of renin
from kidneyhomogenates in a cuvette-based spectrofluorometer.
In Vitro Tissue Imaging of Renin Release: Isolated Microperfused
Tissue, JGA,Renal MedullaMulti-photon fluorescence microscopy
provides deep confocal sectioning of living tissuesin detailed
subcellular resolution with minimal phototoxicity. This ultimately
translatesinto the valuable application of continuous, real-time
imaging to the examination ofintegrated, multicellular
physiological processes. In combination with the in vitro
ex-perimental model, these studies can isolate and examine the
effects of a variable ondefined cellular compartments within living
specimens. This laser-based technology per-mits three-dimensional
imaging, time-lapse studies, and quantitative as well as
qualitativeanalysis. In turn, it offers potential applicability to
the fields of physiology, pharmacology,anatomy, and pathology
within virtually any organ or tissue.
The sensitivity and specificity of multiphoton laser scanning
microscopy (MPLSM) makeit ideal for application to the study of
subcellular structures within thick tissues and even inthe context
of live animals. MPLSM is perfectly suited for optical sectioning
up to severalhundred microns deep into living specimens, offering
ultra-sensitive and quantitativeimaging of organ functions with a
level of temporal-spatial resolution not availablethrough other
imaging modalities. For more than a decade, multiphoton
microscopyhas been successfully paired with various in vitro and in
vivo experimental approachesto study a plethora of different
functions across a variety of organ systems, makingit an
indispensable tool for research. The dynamics of actin filaments,
vesicle release,and the polarity of drug uptake are only some
examples of the phenomena that can beinvestigated. The visual data
obtained provides an unparalleled insight into the
cellularstructurefunction relationships, interactions, feedback
loops, and (patho)physiologicalprocesses at play.
The capabilities of MPLSM have been particularly well harnessed
in studies of light-scattering tissues such as the kidney. The
kidneys are integral to both acute andchronic strategies for blood
pressure and volume homeostasis, utilizing hormonal
(renin-angiotensin system, RAS) as well as structural
(tubuloglomerular feedback, TGF) com-ponents. Using this
experimental approach, such integrated processes may be
visualized,
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Figure 12.12.2 Spectrouorometry readings for ratiometric calcium
signaling (A) and a renin-FRET enzymatic activity assay (B). (A)
Representative recording and procedure of calibratingfura-2
uorescence into absolute values of [Ca2+]. The emission spectrum
collected from 380 nmexcitation (gray) and the intracellular
calcium ratio (black) are used to calculate absolute
concentra-tions according to the equation described in the methods.
(B) The increase in slope shown acrossthe time duration of the
arrow (initial rate) corresponds to an increase in EDANS
uorescence(ANG I production) due to cleavage of the substrate from
renin enzymatic activity.
recorded, and quantified in living tissues or animals at the
cellular, or even subcellular,levels. Protein exocytosis,
intercellular ionic message transmissions, and fluid
transitvelocities may all be captured and measured with this
modality. Experimental interven-tions may be used to instigate
reactions, and real-time videos have the ability to acquirethe
expected results while also uncovering unanticipated reactions to
such stimulation.
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Quantitative imaging of basic renal functions in health and
disease can also providecritical information for characterization
of the delivery and effects of therapeutic efforts.
Multi-photon imaging permits sectioning through an entire
glomerulus (100 m indiameter), and as such has been used
successfully for studies on the isolated microp-erfused afferent
arteriole-glomerulus to examine dynamic processes of
juxtaglomerularstructures. Figure 12.12.3A shows a representative
preparation of a conventional trans-mitted light (DIC) detection
image, clearly depicting the entry of the afferent arterioleinto
the glomerulus. The use of fluorescent probes permits the detection
of cellular andsubcellular structures, and Figure 12.12.3B shows
the same preparation with two-photonfluorescence imaging of
cellular compartments, like renin granules and plasma mem-branes,
in additional detail. With the careful selection of probes, nearly
any cellularmicroenvironment can be examined. Acidotropic
fluorophores including quinacrine andLysoTracker dyes (Invitrogen)
are highly membrane permeant, weakly basic compoundsthat rapidly
accumulate in acidic cellular organelles like renin granules. The
red dye,R18, stains membranes and can be used to delineate the
architecture of vessel walls. Oneof the most commonly used probes,
DAPI, is used to define nuclei as shown in Figure12.12.3C.
The minimal cytotoxicity of multi-photon excitation permits
continuous imaging of livingtissue specimens, and therefore,
real-time imaging of tubuloglomerular feedback (TGF)and renin
release mechanisms are possible. Time-lapse imaging allows the
study of theeffects of various stimuli on the dynamics of renin
release, measured as a reductionof quinacrine fluorescence
intensity during granule exocytosis. An image of a
typicalpreparation superimposed with the field of interest is
illustrated in Figure 12.12.3D. Therelease of renin granular
content (loss of green fluorescence intensity) can be
quantifiedover the course of the process. Not only degranulation,
but also enzymatic activity of thereleased renin (detected as the
generation of angiotensin I) can be visualized in real-timeusing a
FRET-based renin substrate. Together with imaging the actual renin
content,this approach is very useful to monitor the status of the
intra-renal renin-angiotensinsystem, an important target of
anti-hypertensive therapy. In addition to the ability tostudy
integrated processes, the detailed resolution of multiphoton
microscopy permitsthe detection of changes in intracellular
signaling, as shown by the variation in membranepotentials as
assessed by the voltage-sensitive dye, annine-6, in Figure
12.12.3E.
Quantitative Imaging of Kidney Functions In VivoMulti-photon
microscopy has driven many recent advances in the knowledge of
renal(patho)physiological processes: visualization of cellular
variables like cytosolic calciumor pH, cell-to-cell communication
and signal propagation, interstitial fluid flow in the
jux-taglomerular apparatus (JGA), real-time imaging of
tubuloglomerular feedback (TGF)and renin release mechanisms.
Structures below the surface of the kidney in the cortex,such as
the cortical collecting duct and intracellular vesicles, may be
clearly visualized inthe same plane as other structures like the
glomerulus or proximal tubule (Fig. 12.12.4A).The capacity to
simultaneously visualize the glomerulus and proximal as well as
distalsegments of the nephron permits the direct comparison of
structurally connected parts ofthe living kidney. In vivo
quantitative multi-photon imaging can be applied to measure-ments
of many kidney functions, including glomerular filtration and
permeability, concen-tration, dilution, and activity of the
intra-renal renin-angiotensin system. Measurementsof the single
nephron glomerular filtration rate (SNGFR) provide a prime example
of thefunctionally coordinated process that are best visualized and
quantified by the simultane-ous observation of different
structures. An ideal nephron orientation for SNGFR studiesis shown
in Figure 12.12.4B, encountered by selection of the appropriate
plane of interestby scanning across and z-sectioning the kidney.
The acquisition of new visual data has
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Figure 12.12.3 Imaging in vitro preparations microdissected from
mouse kidney. (A) A transmit-ted light differential interference
contrast (DIC) image demonstrating the afferent arteriole (AA)
andattached glomerulus (G). (B) Fluorescence image of an AA-G
preparation. Renin granules (green)are labeled with quinacrine, and
plasma membrane (red) is labeled with R18. (C) Fluorescenceimage of
a glomerulus using the nuclear stain DAPI (blue) and the
membrane-stain TMA-DPH tolabel the podocytes found surrounding
glomerular endothelial cells. (D) DIC image of a glomeruluswith
afferent arteriole (AA) and attached tubule segment (cTAL) for
double perfusion studies. Fluo-rescence image with
quinacrine-stained renin granules (green) is superimposed. (E)
Pseudocolorimage of an in vitro afferent arteriole-glomerulus
preparation stained with the Stark-shift voltagesensitive dye,
annine-6, showing variations in membrane potential in individual
vascular smoothmuscle cells. For a color version of this gure, see
http://www.currentprotocols.com.
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Figure 12.12.4 In vivo imaging and quantication of renal
functional parameters in a Munich-Wistar rat. Quinacrine (green) is
used to label acidic compartments (including renin granules)
and70-kDa rhodamine (red) marks plasma in the intravascular space.
(A) Various cortical segmentsof the nephron may be visualized by
z-sectioning down to 200 m below the surface of thekidney. A
glomerulus (G) opens up into the proximal tubule (PT), and a
collecting duct (CD)lies adjacent. Multiple functional compartments
may be visualized simultaneously, down to thesubcellular level. (B)
The single nephron glomerular ltration rate (SNGFR) may be
measuredtaking xy-t video recordings and calculating the ow of
Lucifer Yellow, an extracellular uid marker,down the early portion
of the PT. (C) In vivo imaging of intracellular pH in the proximal
tubule(PT). BCECF-AM was loaded under the renal capsule in the
living kidney to measure cell pH.Note the primarily apical,
microvillar BCECF uorescence in the PT indicating alkalotic
conditions(bicarbonate reabsorption). For a color version of this
gure, see http://www.currentprotocols.com.
challenged a number of existing paradigms in renal
pathophysiology and ultimately hastremendous promise to provide
non-invasive diagnostic and therapeutic tools in the clinic.
The Munich-Wistar rat strain is an ideal experimental model for
in vivo imaging ofthe JGA and glomerular functions due to its
characteristic superficial glomeruli. Briefly,surgery involves
cannulation of the left femoral artery to monitor systemic blood
pressureand the left femoral vein for fluorescent dye and fluid
infusions. Finally, the left kidneyis exteriorized through a small
dorsal incision and the animal is placed on the stage ofa Leica
inverted microscope with the exposed kidney placed in a
coverslip-bottomedheated chamber bathed in normal saline. Images up
to 200-m deep below the kidneysurface can be collected in
time-series (xyt) with LCS imaging software. The in vivo
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multiphoton model may be used to directly observe and quantify
various(patho)physiological parameters of the kidney including
glomerular filtration rate (GFR)and permeability, blood flow,
tubular flow, urinary concentration/dilution, and renincontent.
Furthermore, integrated and complex functions like TGF-mediated
oscillationsin glomerular filtration and tubular flow may also be
captured. Kidney function maybe quantitatively visualized in health
or disease, including the streptozotocin (STZ)-induced type I
diabetes model. Numerous non-toxic, water-soluble fluorophores can
beused to label specific renal structures, listed in Table 12.12.3.
The circulating plasma orintra-vascular space may be labeled red
with a 70-kDa dextran-rhodamine B conjugate,especially useful for
red blood cell velocity recordings. Tubular segments and,
morespecifically, the content of individual renin granules, can be
visualized using quinacrinein a manner similar to that used in in
vitro applications. The extracellular fluid markerLucifer Yellow
and the gold-standard GFR marker inulin (FITC-conjugated) can be
usedto measure SNGFR. All of these fluorescent probes can be
excited using the same, singleexcitation wavelength of 860 nm
(Mai-Tai), and the emitted, non-descanned fluorescentlight can be
detected by external photomultipliers.
IMPORTANT NOTE: Protocols using live animals must first be
reviewed and approvedby an Institutional Animal Care and Use
Committee (IACUC) or must conform togovernmental regulations
regarding the care and use of laboratory animals.
FLUORESCENCE STUDIES OF CULTURED CELLS: CELL SIGNALINGSTUDIES,
PRIMARY CULTURE, OR CELL LINES
BASICPROTOCOL 1
Cuvette-Based Spectrouorometry to Assess Second-Messenger
Signalingin Living CellsThe following protocol details a method by
which the effect of a stimulus on cellfunction can be investigated
by assessing changes in cellular calcium signaling. A cuvette-based
approach is described here, which uses the cell type of interest
grown on glasscoverslips that is then diagonally inserted into the
cuvette. The cuvette is perfused withvarious solutions with the
help of a pump/vacuum and polyethylene tubing lines in/outof the
cuvette. In this particular system, the emitted fluorescence
(fura-2) is detectedby photometry (counts/sec), but the protocol
can be easily adapted to direct imagingapproaches using camera or
photomultiplier-based fluorescence imaging systems.
NOTE: See Strategic Planning for more detail.
MaterialsCells of interestFura-2 ratiometric calcium imaging dye
(Invitrogen)Dimethyl sulfoxide (DMSO)Krebs Ringer-HCO3 solution
(see recipe)Experimental solutionPBS (APPENDIX
2A)MgCl2CaCl2Ionomycin (membrane permeabilizer)24 40mm glass
coverslipsCuvette-based spectrofluorometer (Quantamaster-8, Photon
Technology) with
heated cuvette holder block (37C) and quartz cuvettesPeristaltic
pump, vacuum, polyethylene tubing for cuvette superfusion and
fluid
exchange
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Load dye into cells of interest1. Grow cells of interest to at
least 75% confluence on 24 40mm coverslips cut in
half lengthwise, which will fit in a standard quartz cuvette
diagonally.
2. Dissolve one vial (50 g) of fura-2 dye in 3 l DMSO and then
dilute into 10 ml ofKrebs Ringer-HCO3 solution, for a final
concentration of 10 M fura-2.
3. Load cells with fura-2 for 30 min (see Support Protocol 1).4.
Wash in 10 ml Krebs Ringer-HCO3 for 20 min at room temperature in
the dark to
remove excess dye.
5. Transfer coverslip to cuvette with 3 ml of Krebs Ringer-HCO3
(37C).6. Perfuse the cuvette containing the cells of interest on
coverslip with Krebs Ringer-
HCO3 for 100 sec to ensure appropriate baseline counts.The ratio
should reach a plateau, indicating cell-bath equilibrium, before
proceedingwith the experiment.
7. Switch perfusate to the experimental solution and continue
recording the change influorescence until the ratio reaches a
plateau.
An increase in the ratio is evidence of an intracellular calcium
release.Quantify changes in calcium signalQuantification of the
intracellular calcium concentrations requires calibration with
thecell membrane permeabilizer, calcium ionophore ionomycin.
Fluorescence intensitiesand ratiometric values in the presence and
absence of calcium will be used to calculateabsolute intracellular
calcium concentrations. The outputs of a calcium signaling studyare
shown in Figure 12.12.2A, and serves as a representation of typical
graphs fromwhich numerical calculations may be made.
8. Prepare 50 ml each of the calibration solutions as
follows:
a. Rmin: PBS containing 10 mM MgCl2 and 2 mM EGTA.b. Rmax: PBS
containing 10 mM MgCl2 and 20 mM CaCl2.
9. Load a new coverslip with fura-2 as in steps 1 to 4.
10. Prepare a 5 mM stock solution of ionomycin dissolved in
DMSO.
11. Dilute 50 l ionomycin stock into 49.95 ml of Rmin to make a
5 M solution ofionomycin dissolved in Rmin.
12. Perfuse coverslip with 5 M ionomycin/Rmin solution for at
least 1000 sec or untilthe ratio plateaus at a minimum.
13. After the Ca2+ ratio has reached a minimal value, switch the
perfusate to 5 Mionomycin/Rmax (50 l ionomycin into 49.95 ml Rmax)
for 500 sec, or until theratio plateaus.
14. To calculate intracellular calcium concentration changes,
use the following formula:
[Ca2+]i = Kd (Sf2/Sb2) [(R Rmin)/(Rmax R)]where, R = ratio
obtained from experiment at alternate 340/380 nm excitation and510
nm emission, Kd = 224 nM, Rmin = value of the minimum Ca2+ ratio
(inabsence of Ca2+), Rmax = value of the maximum Ca2+ ratio (in
presence of Ca2+),Sf2 = value of the counts for 380 nm excitation
in the absence of Ca2+, and Sb2 =value of the counts for 380 nm
excitation in the presence of Ca2+.
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12.12.13Current Protocols in Cytometry Supplement 44
SUPPORTPROTOCOL 1
Enhanced Calcium Loading Techniques with Fura-2 and
Fluo-4Calcium dye loading protocols may need to be optimized for
different cell types. If flu-orescence intensities are
insufficient, dyes may need to be loaded along with reagents
toprevent dye extrusion from cells. For example, to measure
intracellular calcium concen-trations in vascular smooth muscle
cells, cells may be loaded with fura-2 (Invitrogen).Fura-2 is
dissolved in a 20% pluronic acid-DMSO solution and diluted in Krebs
Ringer-HCO3 solution to reach a final concentration of 3M. The dye
is then loaded for 30 min atroom temperature together with 2.5 mM
Probenecid, an organic anion transport blockerthat prevents dye
leakage. Cells are ready for experiments after loading.
A good alternative to fura-2 is the emission ratiometric dye
pair fluo-4/Fura Red. Bothdyes can be excited, e.g., by the argon
laser at 488 nm, but emission is detected in twoseparate channels,
green (fluo-4, peak at 520 20 nm) and red (Fura Red, >600
nm).The fluorophores are diluted to a final concentration of 1 M
and loaded with 250 Msulphinpyrazone to prevent dye leakage for 15
min at room temperature.
BASICPROTOCOL 2
Cuvette-Based Spectrouorometry to Assess Nitric Oxide
ProductionThe appropriate selection of dyes permits the application
of cuvette-based spectrofluo-rometry to studying a countless
variety of second-messenger signals. The Basic Protocoldescribes a
method to study intracellular calcium changes, and the following
protocoloutlines a method for studying the production of nitric
oxide, another important second-messenger. The gas nitric oxide
(NO) is a free radical that diffuses across the plasmamembrane to
affect nearby cells by activating its target, the enzyme guanylate
cyclase,which then produces the second messenger cyclic guanosine
monophosphate (cGMP).Alternatively, NO can also covalently modify
proteins or their metallic cofactors. NOserves many functions,
including the relaxation of blood vessels, the regulation of
neuro-transmitter exocytosis, cellular immunity, and the activation
of apoptosis. It is producedpredominantly from endothelial cells,
neutrophils, and macrophages. The following pro-tocol has been
specifically tailored to work with endothelial cells, but may be
modifiedfor other cell types.
MaterialsEndothelial cells of interestDAF-FM diacetate (nitric
oxide imaging dye; Invitrogen)Dimethyl sulfoxide (DMSO)Krebs
Ringer-HCO3 solution (see recipe), 37CSodium nitroprusside (SNP;
Sigma)Glass coverslipsCuvette-based spectrofluorometer
(Quantamaster-8, Photon Technology) with
heated cuvette holder block (37C) and quartz cuvettesLoad dye1.
Grow endothelial cells to at least 75% confluence on long glass
coverslips.
2. Prepare DAF-FM dye in the dark. Dissolve 50g DAF-FM diacetate
in 10l DMSO,for a 2 M final concentration.
3. Dilute DAF-FM/DMSO into 10 ml of Krebs Ringer-HCO3
solution.
4. Load cells on coverslip with the above 2 M DAF-FM solution
for 10 min at roomtemperature in the dark.
5. Wash cells in 10 ml Krebs Ringer-HCO3 for 15 min at room
temperature in the dark.
6. Transfer coverslip to cuvette with 3 ml of Krebs Ringer-HCO3
(37C).
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12.12.14Supplement 44 Current Protocols in Cytometry
Measure NO production7. Perfuse cells with Krebs Ringer-HCO3
solution (control solution) for 100 sec to
ensure appropriate baseline counts.Signal should gradually decay
in control solution.
8. Switch perfusate to the experimental solution and continue
recording the change influorescence.
An increase in the slope is an indication of the production of
NO.Verify NO productionA positive control experiment is performed
to ensure cell viability and intact NO synthesismachinery. The
cells on coverslip are perfused with the NO donor, SNP, and the
DAF-FMsignal should increase as an indication of NO production.
9. Dissolve 2.9995 mg SNP in 10 ml of Krebs Ringer-HCO3
solution.
10. Add 90 ml of Krebs Ringer-HCO3 solution to make 100 ml total
of a 100 M SNPsolution.
11. Load a new coverslip with DAF-FM as in steps 1 through
4.
12. Perfuse coverslip with 100 M SNP solution for 500 sec,
fluorescence signal shouldbe constantly increasing.
BASICPROTOCOL 3
A NOVEL APPLICATION OF FRET: CUVETTE-BASEDSPECTROFLUOROMETRY TO
EVALUATE CELLULAR ENZYMEACTIVITYThe following protocol details a
method by which renal tissue renin enzyme activity canbe measured
in real-time by using a fluorescent renin substrate. A no-flow
cuvette-basedapproach is described here, which uses all elements of
the enzymatic reaction addedstep-by-step in the cuvette. In this
particular system, the emitted fluorescence (EDANS,fluorogenic
renin substrate) is detected by photometry (counts/sec), but the
protocol canbe easily adapted to direct imaging approaches using
camera or photomultiplier-basedfluorescence imaging systems.
NOTE: See Strategic Planning for more detail.
MaterialsMale mice (for fresh kidney tissue; C57Bl/6, 20 g, 6 to
8 weeks old)Inactin (see recipe)Protease inhibitor (BD
Biosciences)Tissue homogenization buffer (see recipe)Renin assay
buffer (see recipe)Renin-FRET substrate (AnaSpec; see recipe)Tissue
homogenizer (Ultra-Turrax T25 basic, IKA)Orbital shaker1-ml
microcentrifuge tubesCuvette-based spectrofluorometer
(Quantamaster-8, Photon Technology)
Collect kidney cortical homogenate protein1. Sacrifice male mice
(C57Bl/6, 20 g, 6 to 8 weeks old) by Inactin injection
(500 mg/kg b.w. i.p.).2. Remove kidneys and capsule. Slice
kidney into small sections and weigh tissue.
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12.12.15Current Protocols in Cytometry Supplement 44
3. Add tissue to protease inhibitor diluted in homogenization
buffer using the followingparameters:
1 kidney 100 mg = 300 l homogenization buffer and 6 l protease
inhibitor4. Homogenize sample for 2 min at maximum speed using a
tissue homogenizer.
5. Agitate sample for 2 hr at 4C in a cold room on an orbital
shaker at 300 rpm.
6. Transfer lysate to 1-ml microcentrifuge tubes.
7. Centrifuge 20 min at 9300 g, 4C. Collect supernatant.8.
Quantify protein content (by Bradford assay) in each sample to
ensure equal loading
in enzyme activity assays.
Perform renin activity assay in tissue samples9. Warm renin
assay buffer to 37C.
10. Set the spectrofluorometer for assay: excitation at 340 nm,
emission at 490 nm.
11. Add 3 ml of renin assay buffer and 6 l of renin-FRET
substrate to the cuvette.
12. Start baseline reading for 500 sec, during which the signal
should gradually decay.
13. Remove contents of cuvette and repeat step 11.
14. Start reading the fluorescence, pausing after 100 sec to add
homogenized tissuesample (normalized to at least 10 g of protein)
to the cuvette.
The slope of the fluorescence within the first 50 sec of the
addition of the tissue providesan estimate of ANG I
production/renin enzymatic activity.
BASICPROTOCOL 4
IN VITRO TISSUE IMAGING OF RENIN RELEASE: ISOLATEDMICROPERFUSED
TISSUE, JGA, RENAL MEDULLAThe following procedure involves
microdissection of an afferent arteriole-attachedglomerulus
preparation from a sacrificed animal. Alternatively, a preparation
with acortical thick ascending limb of the loop of Henle (cTAL) and
attached glomerulus mayalso be dissected. The preparation is then
placed on the microscope so the focal plane ofinterest is in the
field of view. Fluorescent dyes are loaded by perfusion, and
real-timevideo recordings of any variety of processes, including
renin release, may be acquiredby multiphoton microscopy. An
inverted microscope is useful if the imaging approach iscombined
with micromanipulation of the tissue sample from above (like
microperfusionof dissected blood vessels described in this unit).
Perfusion systems may be customized tofit the needs of the
experiment. Vestavia Scientific provides complete perfusion
systemsas well as interchangeable components (manipulator, stage
plate, perfusion chamber)that are readily applicable for
experiments on tubular structures, like those dissected fromthe
kidneys. Major commercial confocal microscope systems include the
Leica TCSSP5, Zeiss 510 Meta, Olympus Fluoview 1000, etc. Most
microscopes can be poweredby broad-band, femtosecond, fully
automated, infrared (tunable between 700 and1040 nm) combined
photo-diode pump lasers and mode-locked titanium:sapphire
lasers(major brands include the Mai-Tai lasers from Spectra-Physics
and the Chameleon fromCoherent) for multiphoton excitation. For
conventional, one photon-excitation confocalmicroscopy, a variety
of visible and UV lasers are commercially available, e.g., the
redHeNe (633 nm/10 mW), orange HeNe (594 nm/2 mW), green HeNe (543
nm/1.2 mW),and blue Ar (458 nm/5 mW; 476 nm/5 mW; 488 nm/20 mW; 514
nm/20 mW) lasers.The following protocol outlines recommendations
for detecting renin release from theJGA (via changes in quinacrine
fluorescence intensity) when switching from the controlKrebs
Ringer-HCO3 solution to a different solution of interest.
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12.12.16Supplement 44 Current Protocols in Cytometry
MaterialsMice (15 to 20 g)Inactin (see recipe)Dissection medium
(see recipe)Bath medium (see recipe)95% O2/5% CO2 sourceControl
tubular perfusate (see recipe)Krebs Ringer-HCO3 solution (see
recipe)Fluorescent dyes of interest (e. g.,
quinacrine)Thermoregulated Lucite chamber (Vestavia
Scientific)Confocal microscope system (e. g., Leica TCS SP2, Leica
Microsystem)Glass pipets (35-m o. d.)Imaging software (e. g., Leica
LCS)
NOTE: Table 12.12.2 provides a list of fluorophores used to
specifically label variousstructures or fields of interest within
the afferent arteriole-glomerulus complex. Excita-tion/emission
parameters and dye loading recommendations are also included.
Microdissect isolated afferent arteriole-juxtaglomerular
apparatus-glomeruluspreparation1. Anesthetize mice (15 to 20 g)
with Inactin (100 mg/kg b.w. dissolved in water). Cut
renal artery, remove kidney, and sacrifice animal by Inactin
overdose (500 mg/kgb.w. i.p.).
2. Detach renal capsule and store kidney in 5 ml of dissection
medium at 4C. Prepareslices of coronal sections of kidney and
separate medulla. Keep cortex for micro-dissection.
3. Aerate the perfusate/bath medium (a modified Krebs
Ringer-HCO3 solution) with95% O2/5% CO2 for 45 min, and adjust the
pH to 7.4.
Table 12.12.2 Fluorescent Probes Commonly Used in In Vitro
Experiments
Dyea TargetOne photon/multiphotonexcitation (nm)
Loading
TMA-DPH Cell membrane 375/755 1 mM perfusateR18 Cell membrane
543/800 2 mM perfusateHoechst 33342 Nucleus 380/760 2 mM
perfusate/bathQuinacrine Acidic granules (renin) 290/860 5 mM
perfusate/bathLyso Tracker Red Acidic granules (renin) 598 5 mM
perfusateRenin-FRETsubstrate (EDANS)
Renin enzymatic activity 360/720 2 mM bath
Fluo 4 Intracellular calcium 488/850 10 mM perfusate/bathFura
Red Intracellular calcium
( ratio = Ca2+)488/850 10 mM perfusate/bath
DAF-FM Nitric oxide 495 10 mM perfusate/bathBCECF pH ( ratio =
pH) 495/440 10 mM perfusate/bathNile Red Neutral lipids 543/800 2
mM bathaTMA-DPH,
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene
p-toluenesulfonate; EDANS, 5-(2-amino-ethylamino)
nephthalone-1-sulfonic acid. All fluorophores are available from
Molecular Probes, except quinacrine(Sigma) and renin substrate
(DABCYL- -Abu-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS,
AnaSpec).
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12.12.17Current Protocols in Cytometry Supplement 44
4. Dissect a superficial afferent arteriole with glomerulus and
attached distal tubulecontaining the macula densa.
5. Transfer the preparation to a thermoregulated Lucite chamber
mounted onto a Leicainverted microscope.
6. Position the preparation at room temperature so the field of
interest is in the optimalfocal plane.
Load dye and prepare for perfusion7. Load the preparation with
the selected dye to the control arteriolar (or tubular)
perfusate, according to purposes of the study.The loading
conditions must be optimized for each dye, after which they are
removedfrom both lumens. Adding the fluorophore through the
microperfusate offers the advan-tage of much faster loading
compared to incubation with dye in the medium. For
example,perfusion loading with the pH dye BCECF requires only 4 to
5 min to attain the samefluorescence signal/intensity as a 30- to
45-min incubation for cell cultures. The followingsteps outline
recommendations for detecting changes in quinacrine intensity
(renin re-lease) when switching from the control Krebs Ringer-HCO3
solution to a different solutionof interest.
8. Cannulate the afferent arteriole and perfuse with a 35-m o.
d. glass pipet. Maintainperfusion pressure at 50 mmHg (1 psi)
throughout the experiment.
The preparation may also be microdissected with the distal
tubule and macula densa intactfor downstream studies. Cannulate the
distal tubule segment, perfusing at a baseline rateof 2 nl/min.
Tubulo-glomerular feedback may be activated by increasing the rate
oftubular perfusion from baseline at 2 nl/min up to 20 nl/min,
using a constant 10 mM NaClin the perfusate.
9. After cannulation, gradually raise the temperature in the
bath to 37C for the remain-der of the experiment.
10. Continuously aerate the bath with 95% O2/5% CO2.11. Load the
dye through the microperfusate according to the specifications for
each
dye, see Table 12.12.2 for recommendations.For example, in the
case of the acidic granule dye quinacrine, perfuse a 25 M
con-centration for 5 min and then wash out with Krebs Ringer-HCO3
for 30 min to permitstabilization of fluorescent signals.
Image renin release12. Set up the laser settings to the
quinacrine excitation wavelength of 860 nm, and
emission is collected at 510 nm.13. Collect images in
time-series (xyt) with the imaging software (e. g., Leica LCS).
The rate of image acquisition and the duration of study are at
the discretion of theexperiment.
To image renin release, visualized as loss of quinacrine
fluorescence intensity, one frameshould be captured every 10 sec
for 30 min of perfusion with solution of interest.
14. Switch the perfusion solution to the solution of
interest.
15. Collect images in time-series (xyt) for 30 min while
perfusing with solution ofinterest.
16. Measure fluorescence intensity (8 or 12 bit), as well as
vascular and glomerulardiameter values (can be measured with most
imaging software).
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12.12.18Supplement 44 Current Protocols in Cytometry
BASICPROTOCOL 5
QUANTITATIVE IMAGING OF KIDNEY FUNCTIONS IN VIVO BYMULTIPHOTON
EXCITATION LASER SCANNING FLUORESCENCEMICROSCOPYThe methods of red
blood cell velocity and single nephron glomerular filtration
ratemeasurements are briefly described in this protocol. For the
quantitative intravital imagingof other renal functions, see UNIT
12.9. Preparations can be visualized using a two-photonlaser
scanning fluorescence microscope, such as a Leica TCS SP2 AOBS MP
confocalmicroscope system. The Leica LCS imaging software allows
collection of images astime-series videos (xyt) and line-scans
(xt), permitting visualization and quantificationof physiological
function as previously described.
Three water-soluble fluorophores are used to label specific
structures. A 70-kD dextran-rhodamine B conjugate (Invitrogen)
labels the plasma red. Tubular segments and reningranules are
visualized in green with quinacrine (Sigma). Fluorescent probes are
excitedat a wavelength of 860 nm (Mai-Tai) and the emitted
fluorescent light is detected bytwo-channel (red and green)
external photomultipliers.The following is one example of animal
preparation. For alternatives, see the SupportProtocol in UNIT
12.9.
MaterialsC57 BL/6 mice or Munich-Wistar ratsInactin (see
recipe)KetamineKrebs Ringer-HCO3 (see recipe)Fluorescent probes
(70-kDa rhodamine B conjugate, quinacrine, Lucifer
Yellow)Polyethylene tubing (0.86-mm i. d.)Analog single-channel
transducer signal conditioner (World Precision Instruments
model no. BP-1)Two-photon laser scanning fluorescence microscope
(e. g., Leica TCS SP2 AOBS
MP confocal microscope system) and HCX PL APO 63/1.4NA oil
CSobjective lens (Leica)
Perform surgery1. Anesthetize C57 BL/6 mice with Inactin
(thiobutabarbital) at 50 mg/kg and then
inject with Ketamine at 50 mg/kg.Munich-Wistar rats are
anesthetized with Inactin at 120 mg/kg.
2. Cannulate the trachea to facilitate breathing using a piece
of 0.86-mm i. d. polyethy-lene tubing.
3. Cannulate the left femoral vein for fluid and dye infusion
using a piece of 0.86-mmi. d. polyethylene tubing.
4. Cannulate the left femoral artery for systemic blood pressure
measurements using apiece of 0.86-mm i. d. polyethylene tubing and
an analog single-channel transducersignal conditioner.
Calibration can be performed using a pressure manometer model
no. PM-015 and datacan be collected with the data acquisition
system QUAD-161.
5. Make a small left dorsal incision to exteriorize the
kidney.
6. Give bolus injections of dyes through the femoral vein,
avoiding light, and transferanimal to the microscope.
Typically, a red dye like 70-kDa rhodamine B conjugate is used
as a plasma marker andthe green stain quinacrine may be used to
label acidic granules. For rhodamine, 50 l of
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12.12.19Current Protocols in Cytometry Supplement 44
a 10 mg/ml stock dye is diluted in 50 l Krebs Ringer-HCO3. For
quinacrine, 50 l of a25 mg/ml stock dye is diluted in 50 l Krebs
Ringer-HCO3. Each 100-l dye is given bybolus injection and washed
in with 100 l Krebs Ringer-HCO3.
7. Place the animal on the stage of the inverted microscope,
with the kidney in thecoverslip-bottomed heated chamber bathed in
modified Krebs Ringer-HCO3 buffer.
Perform real-time in vivo imaging8. Visualize the kidney from
below using a HCX PL APO 63/1.4NA oil CS objective
lens.High-resolution images can be acquired 150-m deep below the
surface of the renalcortex.
9. Excite the tissue at a wavelength of 860 nm to collect
fluorescence emissions at590 nm for the red rhodamine vascular
signal and 510 nm for the green quinacrineacidic compartment
signal.
10. Move the stage in x, y, or z planes to obtain maximal visual
information from thekidney.
Acquire red blood cell velocity measurementsThe 70-kDa rhodamine
is used as a plasma marker because it is large enough to stayin the
vascular compartment and is not taken up by erythrocytes.
Therefore, the transitof dye-excluding red blood cells can be
captured and measured in repetitive line-scansthrough a central
linear axis of the vessel over time. The motion of red blood
cellscorrelates with the dark bands: distance corresponds to the
movement of the dark bandacross the entire horizontal x-axis and
time corresponds to the vertical y-axis distancetraveled for the
red blood cell to cross the entire field. Red blood cell velocity
ultimatelyprovides an estimate of renal blood flow and is an
important hemodynamic parameter inthe assessment of general renal
function.
11. Estimate renal blood flow by measuring RBC velocity in
cortical capillaries. Takean xt-scan of RBC motion across a
capillary, capturing images with a 1-msec timeresolution.
12. Measure x (distance traveled across the axis) and t (time
elapsed for transit, inthe y-plane). Divide the two variables to
obtain velocity in mm/sec.
For more technical details, see UNIT 12.9. An example of red
blood cell velocity measure-ments, an xt line-scan performed in
renal peritubular capillaries can be found in Figure12.9.3B
Acquire single nephron glomerular filtration rateThe single
nephron glomerular filtration rate (SNGFR) is one possible means of
assessingrenal function. In the same optical plane, a superficial
glomerulus with a clear openinginto a proximal tubule of at least
100 m in length must be selected. The SNGFR is calcu-lated by
measuring the clearance of the extracellular fluid marker, Lucifer
Yellow (LY),whose emission is collected at 528 nm. Using
high-temporal resolution, the fluorescenceintensity changes of LY
are measured at the opening of proximal tubule and downstreamat
least 100 m. The duration of time for peak fluorescence intensity
shifts at proximaland distal regions of interest in the proximal
tubule corresponds to the SNGFR. LY isgiven by bolus i.v.
injection, appears in the glomerulus within 5 sec, and is freely
filteredinto Bowmans space and the early proximal tubule.
13. Take a video (xyt scan) of at least a 10-sec duration.14.
Give a single i.v. bolus infusion of the fluid marker LY into the
femoral vein.
15. Calculate the SNGFR as volume traveled over time.
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12.12.20Supplement 44 Current Protocols in Cytometry
16. Measure the internal diameter, length of the tubule, and
transit time of filtrate betweentwo regions of interest in the
proximal tubule using the Quantify package of the Leicaconfocal
software.
The midpoint of the dye bolus, approximated by the maximal
fluorescence intensity,travels at the same speed as the mean fluid
velocity, so the transit time (shift between theintensity plots at
the two locations) is calculated at the peaks. By calculating
tubular fluidvolume [length (diameter/2)2 ], the absolute value of
SNGFR can be calculated(volume/time).
BASICPROTOCOL 6
IN VIVO IMAGING OF CYTOSOLIC PARAMETERS (Ca2+, pH)In in vitro
model systems, cell cultures and isolated, microperfused renal
tissue techniqueshave been widely used in combination with
fluorescence imaging to measure cytosolic ionconcentrations and
variables including pH, Ca2+, Na+, Cl, cell volume, etc. However,it
may be desirable to confirm if these measurements of cellular
processes are relevantto in vivo conditions that also exist in the
intact kidney. One- or two-photon excitationconfocal imaging,
depending on tissue depth, is ideally suited to achieve this task
withclose to real-time, subcellular resolution. Figure 12.12.4C
demonstrates that it is possibleto measure cytosolic ion
concentrations (e. g., pH) in superficial tubular segments (bothin
proximal and distal tubules) with conventional one-photon
fluorescence excitation.For optical sectioning of deeper
structures, two-photon excitation may be required.Loading of
tubular epithelial cells with a fluorophore is technically the
easiest in thedistal nephron taking advantage of the high luminal
dye concentrations there attainedby the renal concentrating
mechanism. Using mice, a single i.v. bolus injection of thepH
sensitive dye BCECF injected under the renal capsule (Invitrogen,
50 g AM formdissolved in 1 l DMSO and diluted in 50 l Krebs
Ringer-HCO3 solution, excitation at488 nm by the argon laser or at
800 nm by the MP laser, emission at 530 nm) providessufficient
labeling of tubular cells. In preliminary assays in mice, loading
required 5 to10 min, during which time BCECF fluorescence intensity
stabilized at values of at leastone order of magnitude greater than
background fluorescence. Figure 12.12.4C showsthat BCECF
fluorescence intensity was the highest (indicating high, alkalotic
pHi) at
Table 12.12.3 Dyes Used for In Vivo Imaging Studies
Dye Target Laser settings Solution Volume Administration
70-kDadextran-rhodamine B(Invitrogen)
Circulatingplasma/intravascularspace
Ex: 860 nmEm: 590 nm
50 l of a 10 mg/mlstock + 50 lRinger
100 l i.v. bolus
Quinacrine(Sigma)
Acidic granules(renin)
Ex: 860 nmEm: 510 nm
50 l of a 25 mg/mlstock + 50 lRinger
100 l i.v. bolus
Lucifer Yellow(Invitrogen)
Extracellularfluid marker
Ex: 860 nmEm: 528 nm
10 l of a 10 mg/mstock + 90 lRinger
100 l i.v. bolus
BCECF(Invitrogen)
pH Ex: 800 nmEm: 530 nm
50 g dye dissolved in1 l DMSO + 49 lRinger
50 l Injection underrenal capsule
Fluo-4(Invitrogen)
Calcium Ex: 488 nmEm: 520 nm
50 g AM dyedissolved in 1 lDMSO + 49 lRinger
50 l i.v. bolus
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12.12.21Current Protocols in Cytometry Supplement 44
the brush-border membrane region, consistent with significant
bicarbonate reabsorptioninto the relatively small cytosolic volume
of apical microvilli. Developing a reproduciblemethod to measure
cytosolic pH in tubular cells in vivo will be an important tool
todirectly assess the function and activity of ion transport
processes in the nephron and saltand water reabsorption under
various conditions.
To execute this procedure, perform all steps of Basic Protocol 5
with the exception ofstep 6, giving only the 70-kDa rhodamine B and
particular dye of interest (e. g., BCECFor fluo-4) at the
concentrations delineated in Table 12.12.3.
REAGENTS AND SOLUTIONSUse deionized, distilled water in all
recipes and protocol steps. For common stock solutions, seeAPPENDIX
2 A; for suppliers, see SUPPLIERS APPENDIX.Bath medium
Dissolve the following in 100 ml water:1 vial (15.6 g) DMEM/F12
(Sigma)0.12 g NaHCO30.5 g BSAAdjust pH of medium to 7.4 with 1 N
NaoH or 1 N HClAerate with 5% CO2 prior to usePrepare fresh
Control tubular perfusateDissolve the following in 1 liter of
water:0.5844 g NaCl (10 mM)0.3728 g KCl (5 mM)26.352 g
N-methyl-D-glucamine (NMDG)-cyclamate (135 mM)0.1203 g MgSO4 (1
mM)0.2273 g Na2HPO4 (1.6 mM)0.0480 g NaH2PO4 (0.4 mM)6 ml CaCl2
(1.5 mM)0.9008 g D-glucose (5 mM)2.3831 g HEPES (10 mM)Adjust pH to
7.4 with 1 N NaOH or 1 N HClVacuum filter with 0.2-m filter in
sterile hoodWarm to 37C and aerate with 5% CO2 with each useStore
in sterile conditions up to 1 month at 4C
Dissection mediumDissolve the following in 1 liter of water:1
vial DMEM/F121.2 g NaHCO3Adjust pH to 7.4 with 1 N NaOH or 1 N
HClVacuum filter solution with 0.2-m filter in sterile hoodAdd 3%
FBS (30 ml)Heat to 37C and aerate with 5% CO2 prior to each
useStore up to 1 month at 4C
InactinDissolve 120 mg Inactin in 1 ml water. Make fresh for
each use. Inactin is light-
sensitive; keep wrapped in foil until it is used.Inject 0.2 ml
Inactin for a 200 g rat.
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Krebs Ringer-HCO3Dissolve the following into 1 liter of
water:6.7206 g NaCl (115 mM)0.3728 g KCl (5 mM)0.1444 g MgSO4 (1.2
mM)0.0341 g Na2HPO4 (0.24 mM)0.1152 g NaH2PO4 (0.96 mM)0.9909 g
D-glucose (5.5 mM)8 ml CaCl2 (2 mM)2.1003 g NaHCO3 (25 mM)10 g
BSA0.01742 g L-arginine (0.1 mM)Adjust pH to 7.4 with 1 N NaOH or 1
N HClVacuum filter with 0.2-m filter in a sterile hoodWarm to 37C
and aerate with 5% CO2 with each useStore in sterile conditions up
to 1 month at 4CAlways incubate to 37C and pH solution to 7.4 prior
to each use.
For in vivo infusions, supplement Krebs Ringer-HCO3 with BSA
(0.35 g BSA/10 ml KrebsRinger-HCO3).
Renin assay buffer100 mM sodium chloride50 mM Tris baseAdjust pH
to 8.0 with 1 N NaOH or 1 N HClStore up to 6 months at 4C
Renin-FRET substrateDissolve 1.0 mg renin substrate (AnaSpec) in
438.5l DMSO. Store up to 3 months
at 4C in the dark.
Tissue homogenization bufferAdd 20 mM TrisCl and 1 mM EGTA.
Adjust to pH 7.0 with 1 N NaOH or 1 N HCl.
Store up to 6 months at 4C.
COMMENTARYBackground Information
Over the last two decades, confocal micro-scopy and, more
recently, multiphoton micro-scopy, have become fundamental tools
forbiologists and life scientists. Confocal laserscanning
microscopy is a valuable tool forobtaining high-resolution images
and 3-D re-constructions of living biological tissues. Two-photon
excitation is founded on the conceptdeveloped by physicist Maria
Goeppert-Mayerthat two photons of equal energies can com-bine in a
fluorescent molecule to emit a pho-ton of equivalent excitation as
that from theabsorption of a single photon of double the en-ergy.
This synergistic interaction necessitatesthat the two photons
interact nearly simulta-neously with the fluorescent probe nearly
toproduce an emission with a quadratic depen-dence on the
excitation light intensity rather
than the linear dependence of conventionalfluorescence. The
probability of this combina-tory event is extremely low, but
improved byincreasing the number of excitation attemptsmade.
Multiphoton laser scanning microscopywas invented by Watt W. Webb,
WinfriedDenk, and Jim Strickler (Denk et al., 1990).Multiphoton
laser scanning microscopy usessolid state lasers, which emit
photons in100-fsec pulses at a rapid repetition rate(80 MHz) with
longer wavelengths than agas laser. Near-infrared and infrared
light areused for excitation, as the longer wavelengthsallow deeper
penetration into tissues whileavoiding the damaging effects of
conventionalultraviolet or visible illumination on livingsamples.
Briefly, the specimen is illuminatedwith a wavelength twice that of
the absorp-tion peak of the selected fluorophore and the
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12.12.23Current Protocols in Cytometry Supplement 44
combination of two or more photons only oc-curs at the focal
plane, so there is less lightscattering resulting in data with a
more robustsignal-to-background ratio. Electrons outsideof the
focal plane are not sufficiently excitedto fluoresce and cause
bleaching, so the pre-cision of image acquisition does not require
aconfocal pinhole.
The early years following the commercialavailability of
multi-photon microscopy her-alded an era of fascination with in
vivo or-gan imaging. However, interest rapidly shiftedfrom the mere
acquisition of aesthetic im-ages to harnessing the powers of the
technol-ogy for quantitative imaging techniques. Thenext surge of
studies aimed to develop newprocedures or to extend existing
fluorescenceimaging methods to directly observe and quan-tify basic
physiological parameters of the kid-ney including single nephron
glomerular filtra-tion rate (SNGFR), glomerular permeability,blood
flow, tubular flow, tubular reabsorption,urinary
concentration/dilution, renin contentand release, and integrated
functions like thetubuloglomerular feedback
(TGF)-mediatedoscillations in GFR and tubular flow. Multi-photon
techniques have elucidated countlessdynamic processes including
glomerular fil-tration, proximal tubule endocytosis, apopto-sis,
microvascular function, protein expres-sion, and renal cysts at the
subcellular level.Many of these techniques are described inUNIT
12.9.
Two-photon excitation confocal imaging isideally suited to
confirming if measurementsof cellular processes are relevant to,
and reflec-tive of, conditions in the intact kidney. Confo-cal
microscopy also offers the benefits of closeto real-time and
subcellular resolution, whichallows for realistic examination of
physiolog-ical processes. Cytosolic ion concentrations,like pH and
calcium levels, may be measuredin both superficial proximal and
distal tubularsegments with conventional one-photon fluo-rescence
excitation. Two-photon excitation al-lows optical sectioning of
deeper structures.The method has been applied by neurosci-entists
to study ion dynamics in brain slices(Yuste and Denk, 1995) and
even in live ani-mals (Svoboda et al., 1999). Cancer researchhas
utilized the technology for in vivo stud-ies of angiogenesis
(McDonald and Choyke,2003) and metastasis (Wang et al., 2002).
In vitro microperfused tissue modelIntravital multi-photon
microscopy was
used to visualize fenestrations of the afferentarteriole
endothelium in the renin-expressing
segment first described by Rosivall. The workillustrated that
bulk fluid flow in the JGA orig-inated from the afferent arteriolar
ultrafiltra-tion of plasma into the JGA interstitium aswell as the
flow of glomerular filtrate in theBowmans space back into the
extraglomeru-lar mesangium. These studies concluded thatsignificant
fluid flow exists in the JGA, whichmay facilitate filtration of
released renin intothe renal interstitium (endocrine function)
andmay also modulate TGF and renin signals inthe JGA (hemodynamic
function). These find-ings challenge the existing paradigm of
theJGA as a static and isolated microenvironment.
In vivo animal imagingA few aspects of imaging intact organs
in vivo are described here. For more de-tails on intravital
microscopy, see UNIT 12.9.In vivo imaging offers virtually
noninvasiveinsight into live organisms and helps charac-terize
metabolic processes and disease-relatedchanges in the body.
Previously describedmethods have established the use of
multipho-ton imaging for the quantitative and qualitativeevaluation
of various aspects of renal function,including glomerular
filtration and tubular re-absorption (Yu et al., 2005). In vivo
studiesexamine the tissues of whole, living organismswith all
physiological and regulatory compart-ments intact. The approach is
thus ideal fortesting drugs on animals, clinical trials, or
toassess the functional significance of any in-tervention. Although
the experimental modeldoes not isolate potential confounding
compo-nents the way in vitro studies do, the approachis often
preferable for investigating the over-all effects and relevance of
an experimentalvariable on a living subject. Whether the ob-jective
of the study is to compare different in-terventions or to gain
general knowledge about(patho)physiology, the relevance and
influenceof any factor cannot realistically be evaluatedoutside of
the systemic context in which thelocal question resides. Especially
with studiesof the therapeutic efficacy of drugs, it wouldbe
impossible to truly understand the valueof an intervention
independent of potentialmetabolic, regulatory, or compensatory
feed-back systems that may play a role in the livingorganism.
Multiphoton excitation fluorescence mi-croscopy is an excellent
imaging technique fornon-invasive studies of cell, tissue, and
organfunctions in both normal and diseased states.It has the
potential to visualize the delivery,site-specific actions, and
physiological rele-vance of drugs during the development and
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12.12.24Supplement 44 Current Protocols in Cytometry
evaluation of therapies. This technique permitsthe observation,
manipulation, and discoveryof highly complex and integrative
questionsabout the mechanisms underlying pathophys-iological
processes (Sipos et al., 2007). Quan-titative imaging with
multiphoton microscopymay eventually provide a novel
non-invasivediagnostic tool for future clinical applications.For
example, the heterogeneity and decline ofrenal function could be
detected at the ear-liest stages, prior to the onset of
measurableblood and urine signals of underlying pathol-ogy (Kang et
al., 2006a). These innovativetechnologies provide the most complex,
im-mediate, and dynamic portrayal of physiolog-ical function,
clearly depicting and analyzingthe components and mechanisms
involved innormal physiology and pathophysiology.
The combination of two-photon technolo-gies with fluorescent
probes to illuminate spe-cific previously inaccessible tissues,
cells, orintracellular compartments can provide phys-iologically
relevant spatio-temporal informa-tion (Komlosi et al., 2006).
Preserving thestructural architecture and physiological func-tion
of intact tissue is fundamental to studiesof the mechanisms behind
physiological andpathophysiological processes. In addition toits
superlative descriptive power due to highlysensitive imaging of
organ function with ex-traordinary spatial and temporal
resolution,this also translates into the collection of
quan-titative data. Technological advances in micro-scope design,
fluorescent dyes, and analyticalsoftware have synergized to allow
subcellu-lar resolution and simultaneous quantitation ofmultiple
processes. For over a decade, multi-photon microscopy has been
successfully usedwith in vitro and in vivo studies to study
var-ious functions of different organs, includingthe kidney and
lungs (St. Croix et al., 2006).This imaging technology can bring
advancesin the knowledge of pathophysiological pro-cesses across
multiple organ systems. Thus,multiphoton microscopy is particularly
usefulfor in vivo applications where the ability to vi-sualize
events in three dimensions with feed-back mechanisms and humoral
signals intact isabsolutely essential, as in the brain (Garaschuket
al., 2006). New visual and quantitative datamay challenge existing
paradigms in patho-physiology (Rosivall et al., 2006) and havethe
potential to eventually provide novel non-invasive diagnostic and
therapeutic tools forfuture applications. Whether isolating and
re-ducing the field of interest to subcellular mech-anisms, or
investigating complex coordinatedprocesses and their molecular
footprints, mul-
tiphoton imaging offers an unparalleled powerto observe
physiological phenomena and con-textualize their significance.
Critical Parameters andTroubleshootingCells
All of the cuvette-based spectrofluorometryinvestigations of
second-messenger signalinghave analogous experimental designs in
the invitro multiphoton imaging method. The trans-lation of
fluorescence readings into absolutenumbers, as in the case of
ratiometric calciumor pH studies, requires calibration with
cellsfrom the same plate. Renin enzymatic activ-ity experiments can
only be fairly comparedif equivalent amounts of kidney tissue
areadded, therefore, appropriate protein concen-tration
measurements should be performed toensure equivalent loading of
protein betweenexperiments. In some cases, dye washout pe-riods may
vary depending on cell type and up-take capacities. The first 100
sec of spectroflu-orometry readings should thus confirm
appro-priate behavior of cells and dyes in controlsolutions before
proceeding with the experi-ment. All experiments involving
fluorescencedyes must be performed in the dark when pos-sible, with
minimal exposure to light. For bestreproducibility, do not
repetitively freeze andthaw dyes, but rather store in aliquots.
TissueTo ensure the best quality tissue that most
closely approximates living conditions, it isimportant to
dissect the preparation within thefirst hour of sacrificing the
animal and prefer-ably within the first 15 to 30 min if intact
vas-culature is necessary. After the first hour, thetissue begins
losing its physiological tonicity,making the dissections more
difficult to per-form. To preserve structural architecture
andmaintain tissue contents, the tissue must beefficiently
positioned in the chamber and thesuperfusion begun immediately. The
temper-ature of the sample should then be increasedto 37C as soon
as possible after positioningto return the tissue to normal
physiologicalconditions. After ensuring proper pipet posi-tions,
their smooth perfusion should be en-sured because precipitation of
solvents on theglass may sometimes clog the lumen.
Before cannulating the structure of inter-est, there must be no
air bubbles in the perfu-sion pipet. Once the preparation is
perfusing atthe preferred temperature, the glass may moveout from
the focal plane, so the tissue mayneed to be repositioned before
start running the
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12.12.25Current Protocols in Cytometry Supplement 44
experiments. For this reason, acquiring DICimages are important
to verify that fluores-cence signal intensity changes are not an
arti-fact of focal plane changes.
AnimalsThe animals themselves are often the most
irregular variable in these studies. Anesthetiz-ing the animals
sufficiently, but not exces-sively, is important to maintain
physiolog-ical function without eliciting confoundingfeedback
mechanisms. To that end, properbreathing, blood pressure, and
sedation mustbe carefully monitored throughout all parts ofthe
procedure. Keep any fluorescent dyes in thedark to avoid bleaching
and all infusions mustbe at physiological pH and temperature to
en-sure minimal disturbances to homeostatic con-ditions. Scanning
across the renal parenchymaoffers many clues to the condition of
the ani-mal. For example, a collapsed collecting ductsignifies
dehydration and suggests the need forfluid infusion. Technical
complications with invivo animal experiments are minimal if
bloodpressure is monitored and fluid hydration sta-tus maintained.
To avoid unnecessary tissuedamage by the laser, the imageable
surfaceshould be scanned across quickly unless a re-gion of
interest requires further evaluation.
Fluorescent dyesWhen selecting the right fluorophore for a
particular study, the specificity of the probesis an important
issue. The calcium indicatorsfura-2, fluo-4, and Fura Red used in
the pro-tocols all have high affinity and specificityfor calcium.
However, some calcium indica-tors fluorescence can be quenched by
otherdivalent cations like Mg2+ and Mn2+, there-fore, caution must
be used. Visible or infraredlight excited dyes (fluo-4, Fura Red)
are pre-ferred over UV-excitable indicators (like fura-2). When the
use of UV dyes is unavoidable,the fluorescence excitation should be
kept toa minimum (low power, short exposure, longexcitation
intervals) when working with livingcells and tissue.
Probably the most specific indicator for ni-tric oxide has been
DAF-FM, which is ca-pable of detecting NO in low nanomolaramounts.
Other widely used NO indicatorsinclude DAF-2 and
2,3-diaminonaphthalene,which can also detect other free radicals
andreactive oxygen species (ROS), so they are lessspecific for
NO.
For in vivo applications, the fluorophoresmust be non-toxic,
water soluble, and specificfor the organ, cell, or molecular target
of inter-est. For example, the dextran-rhodamine con-
jugates, quinacrine, and Lucifer Yellow listedin the protocols
have been extensively testedand are FDA-approved for human
applications(quinacrine).
Anticipated ResultsAll experiments provide the benefits of
not
only visual information, but also quantifiabledata.
Second-messenger signaling studies canoffer qualitative
confirmation about the pres-ence or absence of an effect.
Furthermore,some specific probes like fura-2 for calciumand BCECF
for pH can be converted into ab-solute numerical values. In vitro
studies withthe same probes offer the same information,with the
added benefits of visual confirmationof the effects and the
opportunity to incorpo-rate the relevance of cell polarity (apical
ver-sus basolateral phenomena) as a component ofthe mechanism.
Furthermore, in vitro investi-gations permit the examination of
multicellu-lar processes and real-time imaging capturesthe time
course in which coordinated eventsare occurring. In vivo studies
incorporate allof the above parameters along with the abil-ity to
contextualize the subcellular phenomenaand physiological
interventions with their rel-evance to systemic functional
measurements(like blood pressure, blood flow, and GFR).Ultimately,
multiphoton imaging offers theunique ability to visualize,
quantify, and com-pare the sequence of events driving
pathophys-iological processes on subcellular and intercel-lular
levels.
Time ConsiderationsCells
Dye loading for cellular studies may takeanywhere between 5 and
30 min, followed bya 20-min wash in Krebs Ringer-HCO3 solu-tion.
Spectrofluorometry experiments to as-sess changes in cellular
signaling moleculesare typically run for 500 sec, although
somestudies may have definitive answers within100 sec.
TissueDye loading for in vitro perfusion studies
is vastly expedited compared to bath loadingfor cells. Dye
loading may take between 5 and10 min, followed by a 5-min wash in
KrebsRinger-HCO3 solution. Real-time imaging ex-periments to assess
changes in fluorescencesignals are typically run for 20 min,
acquiringimages every 5 to 10 sec.
AnimalsAnimal surgeries typically take 30 to
45 min for tracheotomy, arterial and venous
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12.12.26Supplement 44 Current Protocols in Cytometry
cannulations, kidney exteriorization, and dyeinfusion. The
imaging portion of studies ismuch more variable in duration. A
single redblood cell velocity recording takes