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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

Ribonuclease A (RNAse)

Dimethylsulfoxide (DMSO)

Saponin (Sigma)

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

TO-PRO-3 iodide (TP3) (Molecular Probes), note that TP3 requires a flow cytometer capable of red excitation, e.g., 633 nm, 635 nm

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 250 l of PBS supplemented with 2% NCS and 0.1% NaAz and containing 4 g/ml of 7-AAD and mix well.

For staining of cell surface antigen expression add appropriate amounts of FITC-labeled and PE-labeled monoclonal antibodies (mAb) or of FITC and PE isotypic control antibody and incubate the samples while protected from light for 15 min at 20oC - 25oC.

Wash cells once with 2 ml of 1 X PBS by centrifugation at 250 x g for 5 min. Remove the supernatant immediately and completely and add 2 ml of 1 X PBS containing 4 g/ml of AD. Vortex the mixture immediately, spin cells down for at least 5 min at 250 x g, and remove the supernatant completely.

Resuspend cells in 0.5 ml of NASS containing 0.02% of saponin, 4 g/ml of AD, 0.5 M of TP3, and 200 g/ml of RNAse followed by incubation for 30 min at 20oC - 25oC.

If samples were cell surface stained only with PE-labelled mAbs, they are acquired on the flow cytometer in their staining solution after the incubation.

If samples were cell surface labelled with FITC-conjugated mAbs alone or with FITC and PE-labelled mAbs, spin cells down after DNA staining by centrifugation at 250 x g for 5 min; then, resuspend the cell pellet in 0.5 ml of 1 X PBS at pH 7.2 containing 0.02% of saponin, 0.5 M of TP3, and 4 g/ml of AD to restore the FITC fluorescence that is markedly diminished at pH 4.8. Then, acquire samples on the flow cytometer in their staining solution.


Preparation of solutions:

7-AAD stock solution (1mg/ml): dissolve 1 mg of 7-AAD powder first in 50 ml of DMSO, then add 950 ml of 1 X PBS; keep at 4°C 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 H2O 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 4°C.

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 4oC protected from light.