Principle of Flow Cytometry

Transcription

1 Why we use flow cytometer? A powerful system with a simple design. Microscopy qualitative Slow Statistics? 3 Hydrodynamic Focusing Principle of Flow Cytometry 2 Bernas T, Grégori G, Asem EK, Robinson JP. Mol Cell Proteomics Jan;5(1):2-13. Epub 2005 Oct 28. Review. 4

2 Leading Advantages of Accuri C6 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. Bernas T, Grégori G, Asem EK, Robinson JP. Mol Cell Proteomics Jan;5(1):2-13. Epub 2005 Oct 28. Review. 5 Use DI water as sheath fluid CFlow software is easy to learn: zoom in function Compact size, simple maintenance 7 Forward Scatter (FSC) & Side Scatter (SSC) The Accuri C6 is Robust! Side scatter detector Light source Forward scatter detector Lymphocyte Gate National Oceanic and Atmospheric Administration: Great Lakes Microcystis research project Lake Erie The Ecosystem Centre: Palmer Peninsula, Antarctica CD4-PE Gate: CD3 + CD4 + 6 CD3-APC 2 nd Norwich Flow Day: Back of Kate s car, Institute for Food Research 8

3 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 Unique Fluidics System Simplifies Sample Handling 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 o Software: developed by high tech anthropologists trained to facilitate human-computer interactions New and modern approaches exploited wherever possible 9 11 Accuri Innovation - Fluidics Cell Count Verification for the Accuri C6 Waste Laser Purge Sheath Flow Cell Detector Laminar flow fluidics Non-pressurized, peristaltic pumpdriven 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/second Counting Bead: cell count per ml Coefficient of Variation 10,000, ,000, , ,000 y = 1.116x r² = n = 75 direct-volume (C6 Accuri cytometer) C6 counting beads (C6 cytometer) Counting beads p= 0.01 Hemacytometer Hemacytometer p = , ,000 10, ,000 1,000,000 10,000,000 Cell Counting Method Direct Volume: cell count per ml sample Sample 10 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). 12

4 Accuri Innovations in Flow Cytometry C6 optics layout: No dichroics, co-linear lasers 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 Accuri Innovation Pre-optimized and locked-down optics Accuri Innovations in Flow Cytometry Compact optical system design reduces cost and eliminates alignment issues 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) Innovations in all the major components of a flow cytometer o Fluidics: allows direct-volume measurement 488 nm solid state laser 640 nm diode laser PMTs for fluorescence Detection Diodes for scatter detection SSC FSC User changeable optical filters 510/15 540/20 565/20 610/20 780/60 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 16

5 Accuri C6 Detectors are Characterized and Balanced at Manufacture More than 6 logs of dynamic range, 24-bit digital signal processing FL1 = 530/30 FL2 = 585/40 FL3 = 670LP FL4 = 675/25 4 log view 4 log view 8 bit 24 bit 5 log view 18 bit 6 + log view 24 bit 8-Peak Validation Beads (FL1, FL2, FL3) Instrument-to-instrument variation is minimal 6-Peak APC (FL4) Zoom 8-Peak Validation Beads 8-peak data from multiple C6 instruments manufactured over a six-month period. 17 Accuri Innovation Electronics with Broad Dynamic Range A wide range of particle sizes are on scale 18-Bit System 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 Genetics Newcastle University, UK Zoom Area Zoom Area 24-bit Accuri C6 Zoom Area Zoom Area Beads: 1, 2, 12, 29 micron Beads: 1, 2, 12, 29 micron More than 6 decades of signal on a single scale All your data, available at any time 18

6 How does one deal with all that resolution? Accuri Innovations in Flow Cytometry 6 + log view Human peripheral blood HPB Lymph Gate Innovations in all the major components of a flow cytometer o Fluidics: allows direct-volume measurement Zoom Zoom o Optics: locked-down alignment o Signal detection: broad dynamic range obviates voltage adjustments Un-Zoom o Software: developed by high tech anthropologists trained to facilitate human-computer interactions New and modern approaches exploited wherever possible 23 Advantages of Pre-optimzed Detector Settings Pre-Optimized Light Scatter Detectors Adjust Your View with Zoom Greatly reduces risk of lost data due to improper set-up No specialist training or dedicated operator required Zoom Area Predictable, reproducible analysis relative to sample type and application Saves time and sample Attenuation filters (for too-bright signals) give controlled signal reduction Lymphocytes Zoom Again Zoom Out Predictable fluorescence spill-over Focus on the science of measuring fluorescence, not the art of setting voltages 22 24

7 Pre-optimized Light Scatter Detectors Adjust Your View With Zoom Other Modules and Accessories 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 Filters Attenuation filters and optical filters 510/15 540/20 565/20 610/20 780/ CSampler Optional Filters and Selectable Lasers Provide Flexibility Regions Cytometer Status Quads Fluidics Controls Standard Filters YFP GFP YFP Markers Run Criteria FL1: 530/30 Histograms Threshold and Compensation GFP /YFP Combo Dot plots Real Time Updates Plot Statistics 26 Printed with FinePrint trial version - purchase at CD3+CD4+ CD4-APC Density plots CD3-APC-Cy7 GFP FL1: 510/15 CD45RO-PE Sample Grid Selectable Lasers: Reassign laser and detector associations Standard: 488 FL1,2,3 640 FL4 2-blue 2-red: 488 FL1,2 640 FL3,4 4-blue: 488 FL1,2,3,4 Separation of GFP and YFP signals FL2: 540/15 Analysis and Gating Tools FL2: 585/40 Accuri Innovation Intuitive Software CD45RA-FITC CD45-RA - FITC 28

8 Automated Flow Cytometry. Now It s Easy. Define fluorescence spill-over 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 FITC emission intensity 530 BP 585 BP wavelength 4,845 Proportion: Calculate medians 62,421 4,845 62,421 = Screening? Consider the CSampler Pre- and post-compensation 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 30 32

9 Spillover in 1-D: CD45-PE-Cy7 + Spillover not corrected Applications Spillover corrected Predictable Spillover Allows Average Values to be Defined Exploring the Research Power of the C6 Fluorochrome Spillover Slope of graphed data Average Compensation FITC into FL % PE into FL % PE Cy7 into FL % Suggested Compensation Values for the Accuri C6 FITC PE PerCP PerCPCy5.5 PE-Cy7 APC FL1 (530BP) FL2 (585 BP) FL3 (670 LP) Standard 4 color immunofluorescence analysis Cellular proliferation and tracking with cell tracker dyes Cell cycle analysis Multiplex bead analysis: Cytokines Dynamic cell processes: Ca 2+ flux Protein expression and screening: sirna, transfection, infection PE Cy5 into FL % FL4 (675 BP) APC into FL % FSC-A BRDU FITC Bead intensity R2 No virus Luciferase Pyk2 34 CFSE PI - DNA 36 Analyte intensity GFP Expression Printed with FinePrint trial version - purchase at

10 Human PBMC : T cell Phenotyping Gate: CD3+ CD8+ CD8-PE-Cy7 Lymphocyte Gate DNA Detection and Cell Cycle Analysis Data File Format: FCS 3.0 compliant CD3-APC Isotype-FITC CD45RA-FITC Isotype-FITC CD45RA-FITC CD4-PE Gate: CD3+ CD4+ CD3-APC In Vitro T cell proliferation: T cells + dendritic cells (DC) Gate = CD3+ Cells Calcium Flux Measurement with Fluo-4 % of CD3+ T cells Proliferating 0.4% FSC-A T cells only DC + T cells 1:4 Compatible with: FCS Express: CFlow File Importer MultiCycle FlowJo 7.6 WinlistTM and Modfit LTTM VenturiOne 52.4% CFSE 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. Courtesy of: Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI 38 Printed with FinePrint trial version - purchase at 40

11 Multiplex Bead Analysis Assay Designs HSP/Chaperone 8-Plex MultiBead Kit Fluorescent Protein Detection with the Accuri C6 Transfection Screening Inherent Bead Fluorescence (6) (6) Inherent Bead Fluorescence Analyte intensity Analyte intensity 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 Monitor Suppression of Pyk2 Expression by microrna-containing Viruses gp64-pe Expression in SF9 Insect Cells Infected with Baculovirus. Methods: Non-adherent, human PBMCs were stimulated for 3 days with anti- CD3 and anti-cd28 antibodies. The resultant cells were 99% pure for CD4 and CD8 expression (APBT). APBTs were uninfected (No virus/nv) or infected with 2 MOI of a lentivirus that contained GFP and a microrna specific for Lucifierase (luc) or Pyk2 (Pyk2). A. B. No virus Luciferase Pyk2 GFP Expression Virus Generation P2 gp64-pe FSC-H Results: A) The expression of GFP was assessed by flow cytometry. B) The effects of virus infection on Pyk2 and actin expression was assessed by immunoblotting. α-pyk2 α-actin NV Luc Pyk2 P3 Gp64-PE gp64-pe 42 Viral Dilution No virus 1:10 1:100 1:

12 White Cell Differential: Single Platform Cell Counts Simultaneous Analysis of GFP-Transgene Expression and Plant Cell Ploidy FSC vs. SSC CD45 vs. SSC CD45 vs. SSC Granulocytes Eosinophils 2C 4C 8C 16C Image expanded using Zoom tool Monocytes Platelets Lymphocytes Analysis of wild type A. thaliana root nuclei, showing initial PI vs. SSC gate and polyploid populations (2C through 16C). Calculation of cell number per ml of original blood sample for four identified populations. WT GFP Transgenic CD45 vs. SSC Cell Volume Sample Normal Population Gate Number Pulled ( L) Volume ( L) Cells x10 3 /ml Range Lymphocytes P5 60, , Monocytes P6 1, , Granulocytes P7 26, , GFP 2C 4C 8C 16C GFP 2C 4C 8C 16C Analysis of wild type (WT) and GFP transgenic A. thaliana root nuclei. GFPtransgene expression is concentrated in nuclei with greater than 4C DNA content. Eosinophils P3 2, , Log PI Log PI Data courtesy of Dr. David Galbraith, University of Arizona, Department of Plant Sciences, Tucson, AZ, USA DNA Staining with Propidium Iodide Use of 7-AAD for Viability Determination Control (DMSO) HU-331 Viable cells within higher FSC region are impermeable to 7-AAD G 1 Sub - G 1 S G 2 /M Dead cells within lower FSC region are permeable to 7-AAD 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). 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 Pharmingen TM ) per 100 µl volume of cells. 7-AAD Printed with FinePrint trial version - purchase at

13 Absolute counts of viable CD8 and CD4 T cells: Mouse splenocytes C6 Detection of GFP Expression in Bacteria Gate = P1 Light Scatter Gate 7-AAD CD8a PE A Count Volume ( μl) Cells/µL Phenotype Gated on (P1 in all) This Plot 58, Total Cells in P1 Q8-UL 4, Q8-UR Q8-LL 48, Q8-LR 5, Viable CD8 + Cells E. coli B wild type E. coli B + GFP plasmid E. coli B mixture CD8a PE-A Gate = P1 Viable T Cells: Lower Right Quadrant + GFP 7-AAD Count Volume ( μl) Cells/µL Phenotype Gated on (P1 in all) This Plot 58, Total Cells in P1 Q9-UL 4, Q9-UR Q9-LL 45, GFP FL1: 530 BP FL1: 530 BP FL1: 530 BP Q9-LR 7, Viable CD4 + Cells CD4 FITC-A Courtesy of: Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston,TX. 51 Phagocytosis by Macrophages Compensation: why bother? human peripheral blood lymphocytes Macrophages Thymocytes (damaged) Macrophages + Thymocytes CD68 Alexa-647 CD68 Alexa-647 CD68 Alexa-647 TUNEL Alexa-488 TUNEL Alexa-488 TUNEL Alexa-488 Courtesy of James Shayman, M.D. Department of Nephrology, University of Michigan, Ann Arbor, MI, USA 52

14 Advantages of the Accuri C6 innovations Fluidics Non-pressurized Any sample tube Continual calcium flux measurements Absolute cell counting Optics Pre-optimized detectors Fixed optical alignment Reduced training and instrument setup time Reduced maintenance Simpler QC/reduced variability Software Intuitive and easy to use Zoom tool Time as a parameter FCS 3.0 compliant 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 Thank you! Any questions? 54