I got so many requests for the procedure that we here at BDIS decided to post it
on the network for the collective good of all! Please note that this method has
been standardized and used for human PBMC activated with SEB + CD28 for 48 h or
more in order to see significant proliferation. The method will be given in more
detail in a pending manuscript in press - 'BA Mehta and VC Maino. "Simultaneous
detection of DNA synthesis and cytokine production in staphylococcal enterotoxin
B activated CD4+ T lymphocytes by flow cytometry." J Immunol Methods.'
Thanks for all your interest!
Bela.
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Method for BrdU incorporation and simultaneous staining of cell surface markers,
BrdU and cytokines:
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- Add 60 uM BrdU and 10 ug/ml Brefeldin A for the last 6 h of incubation to
activated lymphocytes
- Centrifuge cells, resuspend in 100 ul of FACS Buffer (PBS/0.5% BSA/0.1% sodium
azide) and surface stain using an FL3 dye-conjugated anti-cell surface marker
antibody (eg CD3, CD4 etc. PerCP) for 30 min at RT
- Centrifuge cells, resuspend in FACS Permeabilizing Solution (BDIS, San Jose,
CA); keep at 4 degrees for 18-20 h.
- Wash cells 3x with FACS Buffer, resuspend in 100 ul FACS Buffer for
intracellular staining
- Prepare 10 ug/ml solution of anti-BrdU FITC antibody in 10 mg/ml solution of
DNase in PBS. The DNase solution is to be made FRESH every time, and FILTERED
before use. Add the anti-BrdU FITC at 100-200 ng/test. Add PE-conjugated anti-
cytokine antibodies at the manufacturer's recommended conc. Stain the cells at
RT for 60 min.
- Resuspend in 300-400 ul of 1% PFA and analyze using 3-color flow cytometry.
Note: Some surface markers like CD3, CD4 or CD8 can be stained intracellularly
along with BrdU and cytokines with similar results as cell surface staining.
Saves some time.