Mouse pant isolation kit 2

CD4⁺CD25⁻ T cells were isolated from the spleens of naive C57BL/6 WT mice or CD70^−/− mice using mouse PanT isolation kit II supplemented with biotinylated anti-CD25 and anti-CD8 and LS columns (Miltenyi). With confirmed purify >95%, 1×10⁶ sorted cells were injected via tail veins into gender-matched RAG1^−/− mice, which were weighed initially and then weekly after adoptive transfer. Mice were sacrificed when body weight dropped >20% from initial weight along with signs of diarrhea, hunching, and ruffled fur.

On day -1 WT or CD70^-/- C57BL/6 hosts received 965 cGy from a Cesium-137 source (Mark I, J.L. Shepherd and Associates) at a rate of 114cGy/min. C57BL/6 hosts were transplanted day 0 with BALB/c inoculum as indicated. Mice were weighed twice weekly and considered moribund when body weight reached below 80% of initial weight. In the C57BL/6→BALB/c model, host mice received 792 cGy irradiation on day -1 and were transplanted day 0 with indicated doses of BM + splenocytes or BM + CD25- PanT. T cell depletion was performed using CD90.2 microbeads and LS columns (Miltenyi), resulting in <5% of original T cell composition of BM. PanT and CD25- PanT cells were isolated using Mouse PanT isolation kit II and LS columns (Miltenyi). PanT antibody cocktail was supplemented with biotinylated anti-CD25 for depletion of CD25⁺ cells in CD25- PanT sorts. T cell sorts resulted in >97% purity of desired cell types.