Principle of Flow Cytometry · Accuri Innovations in Flow Cytometry • Innovations in all the major components of a flow cytometer o Fluidics: allows direct-volume measurement o
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®
A powerful system with a simple design.
產品應用專員陳思穎
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Principle of Flow Cytometry
Microscopy
• qualitative• Slow• Statistics?
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Why we use flow cytometer?
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Hydrodynamic Focusing
Bernas T, Grégori G, Asem EK, Robinson JP.Mol Cell Proteomics. 2006 Jan;5(1):2-13. Epub 2005 Oct 28. Review.
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Bernas T, Grégori G, Asem EK, Robinson JP.Mol Cell Proteomics. 2006 Jan;5(1):2-13. Epub 2005 Oct 28. Review.
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Forward Scatter (FSC) & Side Scatter (SSC)
Light source
Side scatter detector
Forward scatter detector
Lymphocyte Gate
CD3-APC
CD
4-PE
Gate:CD3+ CD4+
• Over 7 Decade dynamic range:Eliminating the need to set gain or voltage reduces the consumption of valuable sample and sheath during set-up
• Absolute counts:It is simple to obtain absolute counts, or sample concentration per microliter without the need to add beads to samples
• Automated CSampler®
Standard 96-well (flat, round, and v bottom) Plates, deep-Well 96-well Plates, 48-well Plates, 24-tube rack (for 12x75 mm tube) .<90 minutes for 96-well plate, utilizing 30 second acquisition time per well.
• Use DI water as sheath fluid• CFlow software is easy to learn: zoom in function• Compact size, simple maintenance
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Leading Advantages of Accuri C6
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The Accuri C6 is Robust!
National Oceanic and Atmospheric Administration:Great Lakes Microcystis research project – Lake Erie
2nd Norwich Flow Day:Back of Kate’s car, Institute for Food Research
The Ecosystem Centre:Palmer Peninsula, Antarctica
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Accuri Innovations in Flow Cytometry
• Innovations in all the major components of a flow cytometer
o Fluidics: allows direct-volume measurement
o Optics: locked-down alignment
o Signal detection: broad dynamic range obviates voltage adjustments
o Software: developed by “high tech anthropologists” trained to facilitate human-computer interactions
• New and modern approaches exploited wherever possible
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Accuri Innovation - Fluidics
• Laminar flow fluidics• Non-pressurized, peristaltic pump-
driven system• Patented pulse dampeners• User controls both flow rate and core
diameter• Volume measurement for absolute
counts• Minimum sample volume 25 µL• Up to 10,000 events/secondSheathSheath
PurgePurge
WasteWaste
Flow CellFlow CellLaserLaser
Sam
ple
Sam
ple
Det
ecto
rD
etec
tor
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Unique Fluidics System Simplifies Sample Handling
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• Microprocessor-controlled peristaltic pumps enable direct volume measurement
• Many types of sample tubes may be used• No need to transfer samples • Save time and materials• Add reagents or cells during a run• With CSampler: culture, stain and run, all in one
plate
Cell Count Verification for the Accuri C6
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y = 1.116xr² = 0.9774
n = 75
1,000
10,000
100,000
1,000,000
10,000,000
1,000 10,000 100,000 1,000,000 10,000,000
Cou
ntin
g B
ead:
cel
l cou
nt p
er m
L
Direct Volume: cell count per mL sampleCell Counting Method
1 2 3
Coe
ffici
ent o
f Var
iatio
n
0
10
20
30
40
direct-volume(C6 cytometer)
counting beads(C6 cytometer)
Hemacytometer
Accuri C6 Counting beadsp= 0.01
Hemacytometerp = 0.002
The average coefficient of variation for replicate cell counts using three different counting methods on the same samples.
A paired student’s T test was used to determine p values (95% confidence, N = 23).
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Accuri Innovations in Flow Cytometry
• Innovations in all the major components of a flow cytometer
o Fluidics: allows direct-volume measurement
o Optics: locked-down alignment
o Signal detection: broad dynamic range obviates voltage adjustments
o Software: developed by “high tech anthropologists” trained to facilitate human-computer interactions
• New and modern approaches exploited wherever possible
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FSC
SSC
Accuri Innovation –Pre-optimized and locked-down optics
Compact optical system design reduces cost and
eliminates alignment issues
510/15540/20565/20610/20780/60
User changeable optical filters
488 nm solid state laser
640 nm diode laser
PMTs for fluorescenceDetection
Diodes for scatter detection
FL1 530 / 30nm (FITC/GFP)FL2 585 / 40nm (PE/PI)FL3 >670nm LP (PE-Cy5, PE-Cy5.5, PerCP-Cy5.5, PE-Cy5, PE-Cy7)FL4 675 / 25nm (APC)
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C6 optics layout:No dichroics, co-linear lasers
Accuri Innovations in Flow Cytometry
• Innovations in all the major components of a flow cytometer
o Fluidics: allows direct-volume measurement
o Optics: locked-down alignment
o Signal detection: broad dynamic range obviates voltage adjustments
o Software: developed by “high tech anthropologists” trained to facilitate human-computer interactions
• New and modern approaches exploited wherever possible
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FL4 = 675/25
Instrument-to-instrument variation is minimal
FL3 = 670LP
6-Peak APC (FL4)
8-peak data from multiple C6 instruments manufactured over a six-month period.
Accuri C6 Detectors are Characterized and Balanced at Manufacture
FL1 = 530/30 FL2 = 585/40
8-Peak Validation Beads (FL1, FL2, FL3)
Beads: 1, 2, 12, 29 micron
“A GFP signal that was off scale on our other flow cytometer gave us a distribution that was contained within the 24-bit scale on the C6.”
Ian Dimmick, Flow Cytometry Core Facility Manager, Institute of Human GeneticsNewcastle University, UK
Zoom Area
Zoom Area
Accuri Innovation –Electronics with Broad Dynamic Range
More than 6 decades of signal on a single scaleAll your data, available at any time
18-Bit System
24-bit Accuri C6
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6+ log view
24 bit18 bit
5 log view4 log view
8 bit
8-Peak Validation Beads
More than 6 logs of dynamic range,24-bit digital signal processing
Zoom
4 log view
24 bit
A wide range of particle sizes are on scale
Beads: 1, 2, 12, 29 micron
Zoom Area
Zoom Area
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How does one deal with all that resolution?
6+ log view
Zoom
Human peripheral blood
Zoom
Un-Zoom
HPB Lymph Gate
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Advantages of Pre-optimzed Detector Settings
• Greatly reduces risk of lost data due to improper set-up
• No specialist training or dedicated operator required
• Predictable, reproducible analysis relative to sample type and application
• Saves time and sample
• Attenuation filters (for too-bright signals) give controlled signal reduction
• Predictable fluorescence spill-over
• Focus on the science of measuring fluorescence, not the art of setting voltages
Accuri Innovations in Flow Cytometry
• Innovations in all the major components of a flow cytometer
o Fluidics: allows direct-volume measurement
o Optics: locked-down alignment
o Signal detection: broad dynamic range obviates voltage adjustments
o Software: developed by “high tech anthropologists” trained to facilitate human-computer interactions
• New and modern approaches exploited wherever possible
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Lymphocytes
Zoom Area
Zoom OutZoom Again
Pre-Optimized Light Scatter DetectorsAdjust Your View with Zoom
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Pre-optimized Light Scatter DetectorsAdjust Your View With Zoom
Accuri Innovation – Intuitive Software
Sample Grid
CytometerStatus
Fluidics Controls
Run Criteria
Threshold and Compensation
Real Time Updates
Analysis and Gating Tools
Regions
Quads
Markers
Histograms
Dot plots
Density plots
Plot Statistics
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• Selectable Laser Module
2-blue 2-red: FL1 and FL2 read blue laser-excited emissions;FL3 and FL4 read red laser-excited emissions.
4-blue: All 4 detectors read blue laser-excited emissions.
• CSampler®
• FiltersAttenuation filters and optical filters
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Other Modules and Accessories
510/15540/20565/20610/20780/60
90 %99 %
CSampler®
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Optional Filters and Selectable Lasers Provide Flexibility
Separation of GFP and YFP signals
FL1: 530/30 FL1: 510/15
Standard Filters GFP /YFP Combo
FL2:
585
/40
FL2:
540
/15
GFPYFP
YFP
GFP
Selectable Lasers:Reassign laser and detector associationsStandard: 488 FL1,2,3 640 FL42-blue 2-red: 488 FL1,2 640 FL3,44-blue: 488 FL1,2,3,4
CD3+CD4+
CD3-APC-Cy7
CD
4-A
PC
CD45RA-FITC
CD
45R
O-P
E
CD45-RA - FITC
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• Add-on automation for the C6 Flow Cytometer
• Processes 96-well and 48-well plates
• 24-position rack for 12 x 75 mm tubes
• Priority interrupt for urgent samples
• Easy-to-use software
CSampler®
Automated Flow Cytometry. Now It’s Easy.
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Screening? Consider the CSampler®
• Add-on automation for the C6 Flow Cytometer
• Processes 96-well and 48-well plates• 24-position rack for 12 x 75 mm tubes• Priority interrupt for urgent samples• Easy-to-use software
62,421
4,845
Proportion: 4,84562,421
= .077
Define fluorescence spill-over
FITCemission intensity
wavelength
585 BP530 BP
Calculatemedians
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Pre- and post-compensation
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Spillover in 1-D: CD45-PE-Cy7+
Spillover not corrected
Spillover corrected
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Predictable Spillover Allows Average Values to be Defined
Fluorochrome Spillover
Slope ofgraphed data
Average Compensation
FITC into FL2 0.0770 7.00%
PE into FL1 0.0370 3.50%
PE‐Cy7 into FL4 0.0013 0.00%
PE‐Cy5 into FL4 0.4613 47.00%
APC into FL3 0.0075 1.00%
Suggested Compensation Values for the Accuri C6
FITC PE PerCP PerCPCy5.5 PE-Cy7 APCFL1 (530BP) --- 3.5 0.00 0.0 1.00 ---
FL2 (585 BP) 7.0 --- 0.00 0.00 3.50 0.0
FL3 (670 LP) 1.0 14.5 --- --- --- 1.2
FL4 (675 BP) 0.0 0.0 3.00 12.00 0.00 ---
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Applications
PI - DNAB
RD
U F
ITC
• Standard 4 color immunofluorescence analysis• Cellular proliferation and tracking with cell tracker dyes
• Cell cycle analysis
• Multiplex bead analysis: Cytokines
• Dynamic cell processes: Ca2+ flux
• Protein expression and screening: siRNA, transfection, infection
R2Bea
d in
tens
ity
Analyte intensityCFSE
FSC
-A
Exploring the Research Power of the C6
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No virusLuciferasePyk2
GFP Expression
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Lymphocyte Gate
Human PBMC : T cell Phenotyping
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Isotype-FITC CD45RA-FITC
CD
8-PE
-Cy7
CD3-APC
CD
4-PE
CD3-APC Isotype-FITC CD45RA-FITC
Gate:CD3+ CD8+
Gate:CD3+ CD4+
T cells only 0.4%
DC + T cells1 : 4 52.4%
In Vitro T cell proliferation: T cells + dendritic cells (DC)
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CFSE
Gate = CD3+ Cells% of CD3+
T cellsProliferating
Courtesy of: Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI
FSC
-A
DNA Detection and Cell Cycle Analysis
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Data File Format:• FCS 3.0 compliant
Compatible with:• FCS Express: CFlow File
Importer• MultiCycle• FlowJo 7.6• WinlistTM and Modfit LTTM
• VenturiOne®
Calcium Flux Measurement with Fluo-4
Sample tubes on the C6 do not require pressurization. Agonists can be added during sample acquisition, ensuring that one is able to visualize the entire kinetic activity.
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Multiplex Bead AnalysisAssay Designs HSP/Chaperone 8-Plex MultiBead™ Kit
Methods: Data from Assay Designs™ multiplex bead immunoassay kit for the measurement of HSP client proteins (Akt and Akt pSer473) and HSPs (Hsp27 pSer15, Hsp27 pSer82, Hsp40, Hsp60, Hsp70 and Hsp90 alpha) in cell lysates. Plots show results from a non-heated control and a heat treated hela-2 cell lysate. Standard curves and quantitative analyte levels can be obtained using the MultiBead software provided by Assay Designs.
Inhe
rent
Bea
d Fl
uore
scen
ce
Analyte intensity Analyte intensity
Inhe
rent
Bea
d Fl
uore
scen
ce(6) (6)
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Luc
α-Pyk2
α-actin
Pyk2NV
A.
B.
Monitor Suppression of Pyk2 Expression by microRNA-containing Viruses
Methods: Non-adherent,human PBMCs werestimulated for 3 days with anti-CD3 and anti-CD28 antibodies.The resultant cells were 99%pure for CD4 and CD8expression (APBT). APBTswere uninfected (No virus/NV)or infected with 2 MOI of alentivirus that contained GFPand a microRNA specific forLucifierase (luc) or Pyk2(Pyk2).
Results: A) The expression ofGFP was assessed by flowcytometry. B) The effects ofvirus infection on Pyk2 andactin expression was assessedby immunoblotting.
No virusLuciferase
Pyk2
GFP Expression
Fluorescent Protein Detection with the Accuri C6
Transfection Screening
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gp64-PE Expression in SF9 Insect Cells Infected with Baculovirus.
P2
P3
Virus Generation
ViralDilution
No virus 1:10 1:100 1:1000
gp64
-PE
Gp6
4-PE
gp64
-PE
FSC-H
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White Cell Differential: Single Platform Cell Counts
PopulationCD45 vs. SSC
GateCell
NumberVolume
Pulled (L)Sample
Volume (L) Cells x103/mLNormalRange
Lymphocytes P5 60,827 308.1 2,100 4.15 0.8 - 5.0
Monocytes P6 1,154 308.1 2,100 0.08 0.1 - 1.0
Granulocytes P7 26,618 308.1 2,100 1.81 1.4 - 7.5
Eosinophils P3 2,478 308.1 2,100 0.17 0.0 - 0.4
Calculation of cell number per mL of original blood sample for four identified populations.
FSC vs. SSC CD45 vs. SSC CD45 vs. SSC
Platelets
Lymphocytes
Monocytes
GranulocytesEosinophils
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DNA Staining with Propidium Iodide
Methods: Jurkat cells were treated with either DMSO (Control; <2%) or HU-331, an apoptosis inducing cannabinoid (5 g/mL), Cayman Chemical Company, for 6 hours. Cells were then fixed and stained with Accuri Cell Cycle Phase Determination Kit (KR-300).
Control (DMSO) HU-331
G1
S G2/M
Sub - G1
Simultaneous Analysis of GFP-Transgene Expression and Plant Cell Ploidy
2C
4C8C
16C
Analysis of wild type A. thaliana root nuclei, showing initial PI vs. SSC gate and polyploid populations (2C through 16C).
Data courtesy of Dr. David Galbraith, University of Arizona, Department of Plant Sciences, Tucson, AZ, USA
Analysis of wild type (WT) and GFP transgenic A. thaliana root nuclei. GFP-transgene expression is concentrated in nuclei with greater than 4C DNA content.
GFP
Log PILog PI
GFP
WT GFP Transgenic
2C 4C 8C 16C
2C 4C 8C 16C
Image expanded using Zoom tool
Use of 7-AAD for Viability Determination
7-AAD
Viable cells within higher FSC region are
impermeable to 7-AAD
Methods: Splenocytes from 3 to 6-month old C57BL/6 mice were irradiated with UV light. Cell viability was determined by adding 1 µL 7-AAD from a stock solution of 1 mg/mL (BD PharmingenTM) per 100 µL volume of cells.
Dead cells within lower FSC region are
permeable to 7-AAD
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CD8a PE‐ALight Scatter Gate
Gate = P1
CD8a PE-A
7-A
AD
Viable T Cells: Lower Right Quadrant
Count Volume ( μL) Cells/µL PhenotypeGated on (P1 in all)
This Plot 58,588 6.4 9154 Total Cells in P1Q8-UL 4,354 6.4Q8-UR 243 6.4Q8-LL 48,088 6.4
Q8-LR 5,903 6.4 922 Viable CD8+ Cells
Gate = P1
CD4 FITC-A
7-A
AD Count Volume ( μL) Cells/µL Phenotype
Gated on (P1 in all)This Plot 58,588 6.4 9154 Total Cells in P1Q9-UL 4,495 6.4Q9-UR 571 6.4Q9-LL 45,647 6.4
Q9-LR 7,875 6.4 1230 Viable CD4+ Cells
Absolute counts of viable CD8 and CD4 T cells:Mouse splenocytes
TUNEL Alexa-488
CD
68 A
lexa
-647
Phagocytosis by Macrophages
Macrophages + Thymocytes
Courtesy of James Shayman, M.D. Department of Nephrology, University of Michigan, Ann Arbor, MI, USA
CD
68 A
lexa
-647
TUNEL Alexa-488
Macrophages Thymocytes (damaged)
TUNEL Alexa-488
CD
68 A
lexa
-647
C6 Detection of GFP Expression in Bacteria
Courtesy of: Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston,TX.
+ GFP
- GFP
E. coli B mixtureE. coli B
+ GFP plasmidE. coli B wild type
FL1: 530 BP FL1: 530 BP FL1: 530 BP
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Compensation: why bother?human peripheral blood lymphocytes
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Advantages of the Accuri C6 innovations
Fluidics • Non-pressurized• Any sample tube• Continual calcium flux measurements• Absolute cell counting
Software • Intuitive and easy to use• Zoom tool• Time as a parameter• FCS 3.0 compliant
Optics• Pre-optimized detectors• Fixed optical alignment• Reduced training and instrument setup time• Reduced maintenance • Simpler QC/reduced variability
Electronics• No voltage or gain settings• ALL data collected from every channel all the time• Less sample and time used for instrument setup • Predictable fluorescence spill-over • Simpler QC/reduced variability
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Thank you! Any questions?
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