FLOW CYTOMETRY Not as scary as it sounds!. A BIT OF HOUSEKEEPING…  About me!  About you  Institute  Prior knowledge  Core facility.

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Presentation transcript:

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 µl  Unstained  Isotype control  (Compensation)  Antibody/es d)Fc block

e)Intracellular antigens only  Fix: add 100 µl 4%PFA (30 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 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

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 weren’t 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:  How to present flow data: