To generate stable transfectants expressing Zap70wt or Zap70C564A, P116 cells were transfected with 5–30 µg of pPB[Exp]-Puro-EF1A>hZAP70/T2A/EGFP and 5 μg of pEF_hyPBase. For transient transfections of P116 cells, we used 5–30 µg of the pEYFP-N1-hZap70 vector. DNA electroporation of P116 cells was performed using the Gene Pulser II System (BIORAD) as previously described [16 (link)]. The transfected cells were cultured in an RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. A total of 0.5 μg/mL of puromycin (Gibco, Waltham, MA, USA) was added for the generation of stable transfectants, which were additionally sorted with the Aria Cell Sorter 3 (BD Bioscience) and afterwards maintained in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% Ciprobay.
For the transfection of HEK293T cells, 1 × 106 cells were seeded onto 6-well tissue culture plates one day before transfection. Separately, a mixture was prepared containing 300 μL of 250 mM CaCl2 and 10 μg of pEYFP-N1-hZap70, which was added dropwise under constant agitation to 300 μL of HEPES buffered saline (Sigma-Aldrich). After incubation for 45 min at RT, 3 mL of DMEM (PAN Biotech, Aidenbach, Germany) were added to the mixture, which was subsequently carefully pipetted into the well containing the cells. Cells were incubated in the presence of a transfection medium for 24 h.
For the transfection of HEK293T cells, 1 × 106 cells were seeded onto 6-well tissue culture plates one day before transfection. Separately, a mixture was prepared containing 300 μL of 250 mM CaCl2 and 10 μg of pEYFP-N1-hZap70, which was added dropwise under constant agitation to 300 μL of HEPES buffered saline (Sigma-Aldrich). After incubation for 45 min at RT, 3 mL of DMEM (PAN Biotech, Aidenbach, Germany) were added to the mixture, which was subsequently carefully pipetted into the well containing the cells. Cells were incubated in the presence of a transfection medium for 24 h.
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