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.
Gunzer
M
M
Department of Dermatology, Institute of Cell Biology, University of Muenster, Von Esmarch Strasse 56, D-48149, Münster, Germany. mgunzer@uni-muenster.de
Weishaupt
C
C
Planelles
L
L
Grabbe
S
S
D000818:Animals; D000906:Antibodies; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D002448:Cell Adhesion; D002469:Cell Separation / Q000191:economics / Q000379:methods*; D003362:Cost-Benefit Analysis; D005260:Female; D005434:Flow Cytometry; D018189:Immunomagnetic Separation; D051379:Mice; D008807:Mice, Inbred BALB C; D009757:Nylons; D013154:Spleen / Q000166:cytology* / Q000276:immunology*
D000906:Antibodies; D009757:Nylons