💉人原代T细胞分离培养

实验试剂

实验步骤

人原代T细胞

Human T cells were isolated from whole blood obtained from healthy donors (Saily Bio). In brief,peripheral blood mononuclear cells (PBMCs) were enriched by Ficoll (GE) density centrifugation using SepMate tubes (STEMCELL). CD3+ T cells were isolated from PBMCs using EasySep Human T Cell isolation kit (STEMCELL) and cultured in X-Vivo15 medium (Lonza) supplemented with 5% fetal bovine serum (FBS) (Gibco),human IL-2 (50 ng ml−1,Peprotech),human IL-7 (10 ng ml−1,Peprotech) and 1% penicillin/streptomycin (P/S). To activate T cells,CD3/CD28 dynabeads (Thermo Fisher) were added to the culture at a bead-to-cell ratio of 1:3 for 3 d. After electroporation,T cells were cultured in X-Vivo15 supplemented with 5% FBS and 100 ng ml−1 IL-2.

鼠原代T细胞

CD3+ T cells were isolated from mouse spleen. An individual spleen was homogenized to release splenocytes in 5 ml buffer (PBS + 2% FBS + 1 mM EDTA + 1% P/S). The cell suspension was centrifuged (5 min at 300 g at room temperature) and the cell pellet was resuspended in 1 ml ACK lysis buffer (A1049201,Thermo Fisher) to remove erythrocytes. The single-cell suspension from an individual spleen was pooled,filtered through a 40 μm cell strainer (CORNING) and CD3+ T cells were isolated using EasySep Mouse T Cell isolation kit (STEMCELL). Isolated T cells were activated by culturing in CD3 (2 μg ml−1,Biolegend) and CD28 (2 μg ml−1,Biolegend) coated six-well plates for 24 h. Mouse T cells were cultured in RPMI-1640 (Gibco) medium supplemented with 10% FBS (Gibco),10 ng ml−1 mouse IL-2,50 μM β-mercaptoethanol and 1% P/S (Gibco).