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 murin e 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 u p 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 combina tion with MACS magnetic beads and an autoMACS machine. After purification, cells were viable and mostly non-activated based on the expression of activ ation markers and did not or only minimally respond to polyclonal stimuli s uch 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 suspensio n were recovered at the mentioned purity. The whole procedure is fast: ( < 4 h of preparation), simple and cost effective, as all antibodies and the m agnetic beads have been titrated to the minimal concentration needed for pu rification. 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 aimin g at the physiology of "untouched" murine T cells. (C) 2001 Elsevier Scienc e B.V. All rights reserved.