View source for Staining of cells for flow cytometry

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!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