These washless procedures require proper titration of antibodies.
Procedure A ( frozen cells)
- If adherent, trypsinize and wash once with PBS (without Ca++ and M++, with 2% Bovine Serum Albumin)
- Pellet cells and resuspend in ice-cold Freezing Buffer to a minimum concentration of 105 cells in 50 ml and store at –80°C
- While thawed cells are slowly agitated in an ice bath mounted on a mixer, add 200 ml Lysis-DNA Staining Solution and mix for 15 minutes
- Add 25 ml FITC-conjugated antibody and mix for 30 minutes
- Freezing buffer:
250 mM sucrose
40 mM sodium citrate
5% v/v DMSO
- Lysis-DNA staining solution:
0.5% Nonidet P40
20 mg/ml propidium iodide (PI)
0.2 mg/ml RNAse
0.5 nM EDTA
Procedure B (fresh cells)
- Suspend 2 x 105 fresh cells in PBS and mix with 100 ml Lysing Solution supplemented with appropriate dilution of antibody and incubate 30 min at room temperature.
- Add 100 ml of an appropriate dilution of the secondary FITC-conjugated antibody containing PI (final concentration 10 mg/ml) and RNAse (final concentration 100 mg/ml) and incubate 30 minutes RT.
- Lysing solution:
1% Bovine Serum Albumin (BSA)
0.5% Triton X-100
0.2 mg/ml EDTA
- Larsen, J.K., “Washless” Procedures for Nuclear Antigen Detection. In: Methods in Cell Biology: Flow Cytometry, Vol. 41, Chapter 24, page 377-388. Academic Press, Inc. San Diego. 1994
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