My goal is to establish an in-vitro culture of mouse splenocytes that are lacking in either CD4 T cells or CD8 T cells.
I tried doing this in the past by administering IP injections of anti-CD4 and anti-CD8 depleting antibodies into live mice on Day 1 and again on Day 2 before doing a harvest of splenocytes on Day 3. These splenocytes were then grown in-vitro for four days before analyzing them with flow cytometry on Day 7. However on Day 7 there still appeared to be a population of CD4+ or CD8+ cells showing up in the resulting FACS data.
My question is, did this arise because the depleting Abs were not working? Or could it be the case that CD4/CD8 T cells somehow have the capability to start re-growing again within the in-vitro culture between Day 3 and Day 7? If the latter is the case, are there any methods out there for depleting CD4/CD8 T cells starting with just an in-vitro culture of mouse splenocytes? Thanks!
It will be difficult to deplete all the CD4 T cells or CD8 T cells by using antibody.You can try with some other clones available i but still some cells will persist.The best is to use knockout mice if possible in your study..
The method you have used can not deplete CD4 100%, there are some cells that will persist. The best to use knock out mice, if not try to use positive or negative selection kits for CD4+ cells before doing flowcytometry.
Would you need to deplete CD4 or CD8 T cells in vivo? If not, you could probably deplete CD4 or CD8 cells by FACS or magnetic bead selection following the isolation of splenocytes. In addition, you may use the in vitro depletion methods to check the in vivo depletion efficiency.