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. Scatter profile of lysed whole blood Side Scatter Forward Light Scatter 02004006008001000 0 200 400 600 800 1000 Lymphocytes Monocytes Granulocytes largest and most granular population smallest and least granular population

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

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 Sheath stream Cell Laser beam Flow Cell

9. Cell sorting

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

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 FLUOROCHROMEType of moleculeTypical excitation laserApproximate emission peak Fluorescein isotyocyanate (FITC) Small organic488 nm518 nm AlexaFluor 488Small organic488 nm518 nm Phycoerythrin (PE)Protein488 or 532 nm574 nm PE-Texas RedProtein tandem488 or 532 nm615 nm PE-Cy5Protein tandem488 or 532 nm665 nm Peridinin chlorophyll protein (PerCP) Protein488 or 532 nm676 nm PerCP-Cy5.5Protein tandem488 or 532 nm695 nm PE-Cy7Protein tandem488 or 532 nm776 nm Allophycocyanin (APC)Protein633 nm659 nm AlexaFluor 647Small organic633 nm667 nm AlexaFluor 700Small organic633 nm718 nm APC-Cy7Protein tandem633 nm784 nm Pacific BlueSmall organic405 nm454 nm AmCyanProtein405 nm487 nm

14. Fluorescence spillover Emission of FITC in PE channel

15. Compensating for spillover uncompensated compensated FITC mean fluorescencePE mean fluorescence---------------------------- negativepositivenegativepositive ------------------------------------------ uncompensated12535401851650 compensated1253560135135 1650 - 185 3540 - 125 % 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)