Since I've had several requests for a workable protocol for intracellular
cytoking staining, I'd lot I'd post it for every one.
The procedure I've been using is based on the BD protocol published in
their Application Note 1.
1. Dilute whole blood 1:2 in RPMI 1640
2. Stimulate 1 ml blood in 12 X 75 mm plastic tubes with 25 ng/ml PMA + 1
ug/ml ionomycin in the presence of 10 ug/ml BFA (I always include
an unstimulated control.)
3. Vortex samples briefly and incubate for 4 hrs in 5% CO2 at 37oC
4. Using 100 ul aliquots of sample, add surface antibodies and stain for
15 min at RT (I surface stain with CD8 PerCP and CD3 APC)
5. Add 2 ml FACSLyse, vortex, incubate 10 min in dark
6. Spin 5 min, 500 x g
7. Pour off SN, add 500 ul FACSPerm, vortex, incubate 10 min in dark
8. Wash with 2 ml PBS/BSA/Azide, and spin 5 min, 500 x g
9. Pour off SN
10. Add intracellular antibodies and stain 30 min in dark (I use 5 ul
IFN-g FITC and 10 ul IL-4 PE)
11. Repeat steps 8 and 9
12. Fix in 1% paraformaldehyde.
I then acquire 3000-5000 events in a CD3+ gate and analyze by
quadrant analysis (CellQuest) of dot plots. I calculate % positivity
using BD's formula: (Activ. sample - activ. isotype control) - ( Unstim.
sample - Unstim. isotype control).
I also have a question: I see a more heterogeneous side scatter profile
in the activated cells. How generous should a CD3/ side scatter gate be
to include all relevant events?
-- Michelle
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Michelle N. Fiordalisi, Ph.D.
Clinical Immunology Fellow
University of North Carolina Hospitals
email: mnfiord@med.unc.edu
Phone: 919-966-4058 FAX: 919-966-0486
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