FACS STAINING OF PBLs for monitoring CD4 and CD8 depletion

Wednesday, May 05, 2004

Description
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.

Procedure
Staining Method

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:

a) 50 mcl control (2nd step reagents only)
b) 50 mcl test (CD4 or CD8)
in Falcon 3910 96 well round bottom plates [NB. samples must be contiguous for FACSMate]
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.

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FACS STAINING OF PBLs for monitoring CD4 and CD8 depletion