http://cyto.mednet.ucla.edu/


Materials

Phosphate buffered saline (1 X PBS without Ca++ and Mg++)

Newborn calf serum (NCS)

Sodium azide (NaAz)

Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe

Dimethylsulfoxide (DMSO)

Saponin (Sigma)

7-amino-actinomycin D (7-AAD) stock solution, see recipe

Pyronin Y(G) (PY) (Polysciences)

Actinomycin D (C1) (AD, Roche Molecular Biosystems) stock solution, see recipe

Method

Place 1 x 106 PBS-washed cells into a 12 x 75 mm tube and add 100 l of PBS supplemented with 2% NCS and 0.1% NaAz and mix well.

For staining of cell surface antigen expression add appropriate amounts of biotinylated and allophycocyanin (APC)-labelled monoclonal antibodies (mAb) or of corresponding labelled isotypic control antibody and incubate the samples while protected from light for 15 min at 20C - 25C.

Wash cells once with 2 ml of 1 X PBS by centrifugation at 250 x g for 5 min.

Remove the supernatant and add 100 l of PBSAz containing 2 g of streptavidin Alexa Fluor488R and follow by incubation for 20 min at 4C. Wash cells once with 2 ml of PBSAz by centrifugation at 250 x g for 5 min at 4oC.

Resuspend cells in 0.5 ml of NASS containing 0.02% of saponin and 10 g/ml of 7-AAD followed by incubation for 20 min at 20C - 25C protected from light.

Then, add 1 ml of 1 X PBS and spin cells down by centrifugation at 250 x g for 5 min. Resuspend the cell pellet in 0.5 ml of NASS containing 0.02% of saponin and 10 g/ml of AD and place the mixture on ice protected from light for 5 min.

Add 0.5 l of a 1mg/ml stock solution of PY in distilled water. Vortex immediately and keep on ice protected from light for at least 10 min before sample acquisition on the flow cytometer.

Note: it is possible to keep cells for a maximum of three days protected from light at 4C in the staining solution without adverse effects.

Preparation of solutions:

7-AAD stock solution (1mg/ml): dissolve 1 mg of 7-AAD powder first in 50 l of DMSO, then add 950 l of 1 X PBS; keep at 4C protected from light.

Nucleic acid staining solution (NASS, pH 4.8): 0.15 M NaCl in 0.1 M phosphate-citrate buffer containing 5 mM sodium EDTA and 0.5% BSA fraction V Dissolve 2 tablets of phosphate-citrate buffer in 100 ml of distilled H20 to make a 0.1 M solution.

Add 0.18 g of disodium EDTA to a final concentration of 5 mM.

Add 0.9 g of NaCl to a final concentration of 0.15 M.

Add 0.5 g of BSA to a final concentration of 0.5%. Keep at 4C.

Actinomycin D (AD) stock solution (1mg/ml): dissolve 1 mg of AD powder first in 50 l of DMSO, then add 950 l of 1 X PBS, keep at 4C protected from light.

Save: actinomycin D, 7AAD from manufacturer
http://www.fermentek.co.il/7AAD.htm