A novel method for the purification of sheep red cell rosetting lymphocytes.
A rapid, direct method for the purification of sheep red cell rosetting lymphocytes (ERFC) was developed. The whole procedure, including rosette formation, density separation and hemolysis could be completed within 10 min. A mixture of human peripheral blood mononuclear cells (PBMC) and 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes (EAET) was layered onto Ficoll-Paque without any pretreatment and centrifuged at 600 X g for 2.5 min. The pellet was then immediately treated with an NH4Cl solution containing 10% FCS and hemolysis was completed within 1 min. The purity of ERFC separated in one cycle of the procedure was 98%, the viability 99% and the yield 56% of the initial lymphocyte count. The re-rosetting ability of the prepared cells, after hemolysis, was 95%. The lymphocytes in the fraction prepared by the same method contained 94.3% CD2(OKT11)+ cells, 90% of which were CD3(OKT3)+ cells (T cells) and 9% were CD16(Leu11a)+ cells (NK cells).
Mikamo
S
S
Department of Clinical Oncology, University of Tokyo, Japan.
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D006461:Hemolysis; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*; D012397:Rosette Formation; D012756:Sheep