Two-step negative enrichment of CD4+ and CD8+ T cells from murine spleen via nylon wool adherence and an optimized antibody cocktail.

We developed a method to highly purify CD4+ and CD8+ T cells from murine spleen by negative enrichment strategy. Single-cell suspensions of spleen cells were depleted from erythrocytes by ammonium chloride-mediated lysis. The obtained cell suspension contained approximately 28% CD4+ cells and 14% CD8+ cells. Passing of these cells over a nylon wool column removed up to 75% of all cells, leading to a suspension containing approximately 50% CD4+ and 23% CD8+ cells. These cells were further purified by a single immunomagnetic depletion step using a panel of eight antibodies in combination with MACS magnetic beads and an autoMACS machine. After purification, cells were viable and mostly non-activated based on the expression of activation markers and did not or only minimally respond to polyclonal stimuli such as soluble anti-CD3 antibodies or Concanavalin A. With this method, 19-38% of all CD4 cells and 10-29% of all CD8 cells in a spleen cell suspension were recovered at the mentioned purity. The whole procedure is fast (<4 h of preparation), simple and cost effective, as all antibodies and the magnetic beads have been titrated to the minimal concentration needed for purification. The method is highly reproducible, routinely leading to CD4+ cells with >97% purity (range 97.4-99%) and CD8+ cells with >96% purity (range 95.6-96.7%). The described protocol should facilitate studies aiming at the physiology of "untouched" murine T cells.