Dynal mouse cd4 or cd8 negative isolation kits

Single cell suspensions of donor strain splenocytes were prepared as described (21 (link)) and transferred into age matched BDF1 hosts by tail vein injection. The percentages of donor CD4 and CD8 T cells populations were analyzed by flow cytometry and donor inoculum adjusted prior to transfer to achieve the desired amount of donor CD4 and CD8 T cells as indicated in the text and/or respective figure legends. In some experiments, acute GVHD was induced using purified donor T cell subsets achieved through negative isolation using Dynal mouse CD4 or CD8 negative isolation kits (Invitrogen, Carlsbad, CA) which deplete B cells, NK cells, monocytes/macrophages, dendritic cells, granulocytes, platelets, erythrocytes and either CD8 or CD4 respectively. Purity was confirmed by flow cytometry and was typically > 95%.

Single cell suspensions of donor strain splenocytes were prepared as described [17 (link)] and transferred into age and sex matched BDF1 hosts by tail vein injection. The percentages of donor CD4⁺ and CD8⁺ T cells populations were analyzed by flow cytometry and donor inoculum adjusted prior to transfer such that all F1 mice received the number of donor CD4 and CD8 T cells designated in the Figure Legends. For Figs 1-4 this typically required 55-65 × 10⁶ unfractionated donor splenocytes. In Fig. 5, GVHD was induced using purified donor T cell subsets achieved through negative selection using Dynal mouse CD4 or CD8 negative isolation kits (Invitrogen, Carlsbad, CA) which deplete B cells, NK cells, monocytes/macrophages, dendritic cells, granulocytes, platelets, erythrocytes and either CD8 or CD4 respectively. Purity of the donor inocula was confirmed by flow cytometry and was typically > 95%.