Staining of cells for flow cytometry

Materials
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