® A powerful system with a simple design. 產品應用專員 陳思穎 2 Principle of Flow Cytometry Microscopy • qualitative • Slow • Statistics? 3 Why we use flow cytometer? 4 Hydrodynamic Focusing Bernas T, Grégori G, Asem EK, Robinson JP. Mol Cell Proteomics. 2006 Jan;5(1):2-13. Epub 2005 Oct 28. Review. Printed with FinePrint trial version - purchase at www.fineprint.com
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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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• 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
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
• 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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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.
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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