This method is useful for monitoring depletion in vivo using rat IgG2b monoclonal antibodies against CD4 and CD8. The indirect staining method, using a monoclonal reagent that does not give any background on mouse Ig, will pick up any residual CD4 or CD8 T-cells still coated with rat IgG2b antibodies. The optional second colour staining for CD3 allows more accurate enumeration of individual T cells subsets, and checks for any CD4 and CD8 double negative T cells. It is also possible to add a fixed number of 10 micron beads (eg 10E5 per 200mcl of blood) that can be visualised and counted in a separate forward and side scatter gate on the FACScan in order to estimate absolute numbers of each subpopulation.
Note: mcl is short for microlitres
1. Take 1-200mcl of blood into 1 mcl heparin (ie. 1U) in microfuge tube
2. Add 0.9ml water for 10 seconds - this must be at room temperature (RT=20-25 deg C)
3. Add 0.1ml 10x phosphate buffered saline (PBS), mix well and spin at 1200rpm for 5 minutes (RT).
4. Carefully remove supernatant and RBC ghosts leaving WBC pellet (difficult to visualise at this stage).
5. Repeat steps 1-4 above, should now be able to see WBC pellet (just).
6. Suspend in 100mcl of cold block buffer (PBS x 1, BSA 1%, 5% HINRS [heat inactivated normal rabbit serum], 0.1% sodium azide.)
7. Split 6 above into:
8. Add 50mcl of rat IgG2b anti-mouse CD4 (YTS 191.1) or anti-CD8 (YTS 169.4) as either neat supernatant or 20mcg/ml mAb in block buffer for 1 hour at 4 deg C. [For optional 2 colour staining also include rat IgG2a anti-CD3-biotin (eg. KT3-biotin) at 10mcg/ml].
9. Wash x 2 with 200mcl PBS, 0.1% BSA, 0.1% sodium azide (wash buffer), spinning the cells down using plate carriers at 1000 rpm for 2 minutes each, and then sucking off the supernatant.
10. Add 50mcl of FITC-monoclonal anti-rat IgG2b (10mcg/ml NORIG-7.16-FITC from Serotec, diluted in block buffer) to the cell pellet, resuspend and incubate for 1 hour at 4 deg C (for optional 2 colour staining also include Streptavidin-R-Phycoerythrin [SAPE from Sigma diluted 1/20], although it may then be advisable to leave out HINRS from block buffer as this may contain biotin).
11. Wash once with 200mcl wash buffer (or IMDM 0.1% BSA 0.1% sodium azide [contains biotin to block free sites on Streptavidin and avoid agglutination/quenching] for two colour staining).
12. Wash once more with 200mcl wash buffer.
13. Add to pellet 50mcl 1% BSA in PBS and resuspend well.
14. Add 50mcl 4% formalin in PBS, mix, cover in foil and store at 4 deg C until ready to run on FACScan.
Steve Cobbold 31/7/96