Requirements for any cell separation
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 to be separated
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
Basic Sorting Buffer
1x PBS (Ca/Mg++ free)-1mM EDTA-25mM HEPES pH 7-0.5% Fetal Bovine Serum (heat inactivated)+ 1% antibiotics (penicillin and streptomycin)
1x PBS (Ca/Mg++ free)
Up to 50 mL
2.5mM EDTA (stock 500mM)
25mM HEPES pH 7.0 (stock 1M)
0.5% FBS (Inactivated)
1% antibiotics (pen and strep)
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.
Optimum concentration of cells
Nozzle orifice size
Final concentration (per mL)*
8-15 x 106
Activated cells, small cell lines
7-10 x 106
Adherent cells, large cells
5-9 x 106