Anatomy of a flow cytometer

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2 Anatomy of a flow cytometer Fluidics Optics Electronics Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered, and converted to digital values that are stored on a computer

3 Fluorescence Detectors If the cell is labeled with a fluorescent probe which is excited by the laser, the cell will emit fluorescence as it passes through the laser beam. Increasing Fluorescence

4 Cell-by-Cell Analysis Fluorescent Plate Reader Flow Cytometer Cell # FSC SSC FL1-H FL2-H FL3-H ,

5 Data Display -Histograms offscale data 1024 sets Frequency Distribution

6 Light Scatter Size Shape Granularity Forward Scatter (FSC, FALS) Most sensitive to size & collection angle Side Scatter (SSC, 90LS) Most sensitive to granularity

7 Peripheral Blood Light Scatter Lymphocytes- small, little complexity Monoctyes-larger, more granular Neutrophils-large, lots of granules

8 FluorochromeComparisons Comparative Intensities of CD8 Conjugates FITC PE ECD PC5 PECy5.5 PC7 Pacific Blue APC Alexa 700 APCAlexa 750 Pacific Orange Intrinsic Characteristics Extinction Coefficient Quantum Yield Emission Spectral Overlap Instrument Optics Filters PMT Sensitivity Laser wavelength & power

9 FluorochromeSelection FITC isotype PE isotype 18% 71% E-Cadherin FITC E-Cadherin PE Low density expression needs a bright fluorochrome.

10 Compensation Correction for spectral overlap FITC spillover compensated FITC & PE Emission Spectra FITC only sample

11 Compensation Rules of the Road 1.Controls need to be as bright or brighter than any sample that the compensation will be applied to. 2.Background fluorescence should be the same for the positive and the negative control. 3.Compensation controls MUST match the exact experimental fluorochrome.

12 Analysis-6 colors

13 Analysis 6 colors, 15 dot plots

14 MultiparameterAnalysis 6 colors Data filtering to select populations of interest Color events based on gates to visualize multiparameter data

15 Cell Sorting presort postsort Sort Purification Subset of interest = 1% Sort rate = 20,000 total cells/s Collection rate = 200 cells/s Or ~83 min for 1 million cells Walter and Eliza Hall Institute Flow Cytometryhttp://

16 Plate Sorts Sort into any size plate with any number of cells per well

17 Tips for better cell sorting Avoid cell aggregates Use a viability stain Bright markers yield higher purity Avoid compensation as much as possible J. Dow, NC State Vet Med

18 Our favorite websites Flow Cytometry- A Basic Introduction, Michael Ormerod Compensation, Mario Roederer Life Technologies Fluorescence Tutorials Spectraviewer University of Chicago Flow Cytometry Blog Spot, Ryan Duggan 10 steps to a successful flow cytometry experiment Antibody titrations What is MFI?

19 Submitted Questions I m new to the Gallios. How do I choose voltage settings? Use the minimum voltage that resolves a dim population Determine using the CV of unstained cells or signal:noise of stained cells Brightest positive signals must be on-scale PMT settings should be balanced to permit compensation PMT Filter Suggested starting voltage 1 525/ / / / LP / / LP / /40 450

20 Submitted Questions Why am I having trouble analyzing Gallios data with FlowJo? Scaling problems Compensation problems Scaling problems seem to be fixable, but compensation should be done on the Gallios Kaluza for Gallios acquisition data (FCS3.1) should work with FlowJoMac version 8 or better FlowJo X

21 Submitted Questions What do I need to think about for quality control of flow assays? Instrument QC Alignment check-%cv PMT standardization- adjust voltage to place reference bead in same channel Check compensation Antibody QC Titer new lot of antibodies Check for lot-to-lot consistency Check compensation Reagent QC Validate new lot of reagents (Buffers, lyse reagents)