Basics of Flow Cytometry

1. Basics of Flow Cytometry Holden Maecker

2. Outline • Definitions, what can be measured by flow cytometry • Fluidics: Sheath and sample streams, flow cells, sorting • Optics: Lasers, filters • Electronics: PMTs, signal processing • Fluorochromes: spectra, spillover • Data analysis: FSC files, gating, statistics

3. Definitions • Flow cytometry: study of cells as they move in fluid suspension, allowing multiple measurements to be made per cell. • FACS™: fluorescence-activated cell sorting

4. What measurements can be made? • Forward light scatter (FSC): proportional to cell size • Side light scatter (SSC): proportional to cell granularity • Fluorescence: • Binding of fluorescent-labeled antibodies • Ca++-sensitive dyes within cells • Fluorescent proteins expressed by cells • Binding of DNA dyes

5. largest and most granular population 1000 Granulocytes 800 600 400 Monocytes 200 smallest and least granular population Lymphocytes 0 0 200 400 600 800 1000 Scatter profile of lysed whole blood Side Scatter Forward Light Scatter

6. Fluorescence data display Negative control histogram PE Number of Events FITC Fluorescent Intensity  FITC

7. Major components of a flow cytometer • Sample intake port • Sheath and waste reservoirs • Flow cell • Laser(s) • Optical filters • PMTs (photomultiplier tubes) or photodiodes • Signal processor

8. Cytometer fluidics create laminar flow Sample stream Flow Cell Sheath stream Laser beam Cell

9. Cell sorting

10. Typical 2-color cytometer configuration FL1 PMT 488/10 nm band pass filter 530/30 nm band pass filter SSC PMT 1% ND front surface mirror FL2 PMT 560nm short pass dichroic mirror 585/42 nm band pass filter 488nm band pass filter 488nm laser beam FSC PD flow cell

11. Background and autofluorescence • All cells have a certain level of background fluorescence, due to: • Autofluroescence: from pigments and fluorescent moieties on cellular proteins • Non-specifically bound antibodies, and free antibody in the sample stream • The level of autofluorescence varies with the wavelength of excitation and collection: • Highest in FITC, PE detectors; lowest in far red (APC, Cy7) detectors

12. Fluorescence sensitivity • Detection Efficiency (Q): number of photoelectrons generated per molecule of fluorophore • Dependent upon fluorophore, filters, PMT sensitivity, voltage gain setting, etc. • Background (B): non-specific signal intrinsic to the system • Dependent upon autofluorescence, unbound fluorophore, stray light, etc.

13. Common fluorophores for Ab conjugation

14. Fluorescence spillover Emission of FITC in PE channel

15. 1650 - 185 • 3540 - 125 Compensating for spillover compensated uncompensated FITC mean fluorescence PE mean fluorescence ---------------------------- ---------------------------- negative positive negative positive ----------- ---------- ----------- ---------- uncompensated 125 3540 185 1650 compensated 125 3560 135135 % Spillover = X 100

16. FCS files • FCS 2.0 and FCS 3.0 conventions • Often referred to as list-mode files • Contain all of the measurements (FSC-H, FSC-A, SSC-H, SSC-A, FL1-H…) for each individual cell processed in a given sample

17. Hierarchical gating

18. Web reference tools • BD Spectrum Viewer: http://www.bdbiosciences.com/spectra • Maecker lab weblog: http://maeckerlab.typepad.com (protocols, manuscripts, literature updates)