FLOW CYTOMETRY Not as scary as it sounds! A BIT OF HOUSEKEEPING About me! About you Institute Prior knowledge Core facility OVERVIEW Flow = fluid Cyto = cell Metry = measurement FORWARD SCATTER (FSC) SIDE SCATTER (SSC) FLUORESCENT MARKERS Size (FSC) Internal complexity (granularity) (SSC) Fluorescence intensity (relative expression of marker) { SAMPLE PREPARATION: PREWORK Choose your antibodies How many? Which fluorochromes? Will they need compensation? Which machine to use? BD FACSCANTO II FORTESSA X20 Compensation? Nooooooo Undercompensated Overcompensated Correctly compensated SAMPLE PREPARATION: STAINING a)Prepare FACS buffer b)Detach cells surface receptor? c)Resuspend cells min. 200,000 cells/tube, max 100 l Unstained Isotype control (Compensation) Antibody/es d)Fc block e)Intracellular antigens only Fix: add 100 l 4%PFA (30 RT) 2x 1ml FACS wash Perm: 2x 2ml Permeabilisation buffer wash Resuspend 100 l Perm buffer f)Antibody staining Add 10 l Ab/10 6 cells Incubate 1h in the 4C (surface) or RT (intracellular) Wash 2x FACS buffer (surface) or 1x Perm buffer + 1x FACS buffer Resuspend in 200 l FACS buffer g)(Beads) h)Analyse DATA ACQUISITION & ANALYSIS a)Set up experiment b)Acquire cells PE positive APC negative PE positive APC positive PE negative APC positive WHAT HAPPENED HERE? A) All cells are dead! B) Some of my cells are doublets Height Width Cells are growing / T-cell activation C) My cells are not displayed! D) Unstained and isotype werent fixed & permeabilised E) Cells are autofluorescent THIS IS BUT A SCRATCH Cell identification Population sorting Cell proliferation Cell death Functional measurements (pH, Calcium) And more THANK YOU! Questions? INTERESTED IN MORE? Theory behind flow: Design your experiment: https://fluorofinder.com/panels/newhttps://fluorofinder.com/panels/new How to present flow data: