Negative control: unlabeled cells and cells labeled with isotype control (for GFP parental cells).

Compensation controls if necessary: If staining is done with several fluorochromes, monolabeling of each fluorochrome will be necessary.

The cell death label will be added by the Flow Cytometry Service and its choice will be depend on the combination of fluorochromes present in the sample.

The controls only need a final volume of 500 µl with 500,000 cells. They can be brought in FACS tubes in FACS Buffer or Sorting Buffer.

The cells should be stored at 4ºC and protected from light.

Samples in sterile tubes in ice.

They should always be filtered with a cell strainer.

The concentration should be 30 x 106 cells/ml. When the cells are larger than 20 microns, the concentration should be around 10 x 106 cells/ml.

In 15-ml culture tubes, put 1 ml culture medium containing 20% serum or sterile FBS. You can calculate the approximate volume of cells you are going to collect and use smaller tubes (FACS, Eppendorf tubes); if you wish, check with the Flow Cytometry Service.

Cytometry tubes: Ref. 352052 BD Falcon

15 ml polypropylene tube: Ref. 430791 Corning or similar

50 micron (50 u) cell strainer: Ref. 352350 BD Falcon

0.5M EDTA, pH 8.0 (4 x 100 ml): Ref. 15575-020 GIBCO

Cytometry tube with cell strainer cap: Ref. 352235 BD Falcon

1x PBS (Ca/Mg++ free)-1mM EDTA-25mM HEPES pH 7-0.5% Fetal Bovine Serum (heat inactivated)+ 1% antibiotics (penicillin and streptomycin)

Store at 4ºC

If the cells are adherent, the concentration of EDTA should be increased to 5 mM.

If there is a large number of dead cells, adding 10U/mL DNAase II is recommended.