1. FLOW CYTOMETRY Not as scary as it sounds!

2. A BIT OF HOUSEKEEPING…  About me!  About you  Institute  Prior knowledge  Core facility

3. OVERVIEW Flow = fluid Cyto = cell Metry = measurement

5. FORWARD SCATTER (FSC)

6. SIDE SCATTER (SSC)

7. FLUORESCENT MARKERS

8. Size (FSC) Internal complexity (“granularity”) (SSC) Fluorescence intensity (“relative expression of marker”) {

9. SAMPLE PREPARATION: PREWORK Choose your antibodies  How many?  Which fluorochromes?  Will they need compensation?  Which machine to use?

10. BD FACSCANTO II FORTESSA X20

11. Compensation? Nooooooo…

12. Undercompensated Overcompensated Correctly compensated

13. SAMPLE PREPARATION: STAINING a)Prepare FACS buffer b)Detach cells  surface receptor? c)Resuspend cells  min. 200,000 cells/tube, max 100 µl  Unstained  Isotype control  (Compensation)  Antibody/es d)Fc block

14. e)Intracellular antigens only  Fix: add 100 µl 4%PFA (30 min @ RT)  2x 1ml FACS wash  Perm: 2x 2ml Permeabilisation buffer wash  Resuspend 100 µl Perm buffer f)Antibody staining  Add 10 µl Ab/10 6 cells  Incubate 1h in the dark @ 4C (surface) or RT (intracellular)  Wash 2x FACS buffer (surface) or 1x Perm buffer + 1x FACS buffer  Resuspend in 200 µl FACS buffer g)(Beads) h)Analyse

15. DATA ACQUISITION & ANALYSIS a)Set up experiment b)Acquire cells

16. PE positive APC negative PE positive APC positive PE negative APC positive

17. WHAT HAPPENED HERE? A) All cells are dead!

18. B) Some of my cells are doublets Height Width Cells are growing / T-cell activation

19. C) My cells are not displayed!

20. D) Unstained and isotype weren’t fixed & permeabilised

21. E) Cells are autofluorescent

22. THIS IS BUT A SCRATCH… Cell identification Population sorting Cell proliferation Cell death Functional measurements (pH, Calcium) And more…

23. THANK YOU! Questions?

24. INTERESTED IN MORE?  Theory behind flow: http://www.bdbiosciences.com/eu/s/training/e-learninghttp://www.bdbiosciences.com/eu/s/training/e-learning  Design your experiment: https://fluorofinder.com/panels/newhttps://fluorofinder.com/panels/new http://m.bdbiosciences.com/us/s/spectrumviewer  How to present flow data: http://www.ncbi.nlm.nih.gov/pubmed/18752282http://www.ncbi.nlm.nih.gov/pubmed/18752282