BD fastimmune protocol

Michelle N Fiordalisi mnfiord at
Wed Feb 26 09:39:08 EST 1997

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

Michelle N. Fiordalisi, Ph.D.
Clinical Immunology Fellow
University of North Carolina Hospitals
email: mnfiord at
Phone: 919-966-4058  FAX: 919-966-0486

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