Staining of cells for flow cytometry

FACS buffer:	1xPBS FCS (heat-inactivated, final concentration: 5%) 5% Natriumazid (final concentration: 0.1%)
1st antibody:	primary antibody (approx. dilution 1:200)
2nd antibody:	secondary antibody (approx. dilution 1:500) fluorescence- labelled
controls:	sample with irrelevant primary antibody and secondary antibody sample with secondary antibody only if possible untransfected cells stained with both antibodies

Procedure

Preparation of cells:

§   aspirate medium and wash twice with 5-10 mL 1xPBS

§   detach cells with 1 mL Trypsin/EDTA

§   inactivate Trypsin/EDTA with 3 mL fresh medium

§   transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm

§   discard supernatant and resuspend the cell pellet in FACS buffer

§   centrifuge again, resuspend cells in FACS buffer and determine cell count

§   distribute into Eppendorf tubes (106 cells/tube)

Staining (perform all steps on ice):

§   pellet the cells by centrifugation 2 min, 2500 rpm, 4°C

§   resuspend pelled in 300 µL FACS buffer

§   add 1st antibody at desired concentration

§   incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C

§   discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above

§   wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer

§   add 2nd antibody at desired concentration

§   incubate max. 30 min on ice (keep samples dark!)

§   wash twice with 600 µL FACS buffer

§   during centrifugation prepare FACS tubes with 600 µL FACS buffer

§   resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes

§   measure samples