All chemicals and cell culture supplies were from Sigma if not otherwise noted. Primary murine T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 kU/ml penicillin-streptomycin, 50 μM β-mercaptoethanol and 1 mM sodium pyruvate. The phoenix packaging cell line for retroviral infections was cultured in DMEM medium supplemented with 10 % FBS, 2 mM L-glutamine, 1 kU/ml penicillin-streptomycin. All cells were grown in a humidified atmosphere at 37 °C and 5% CO2.
For expression of the CD3ζ-PS-CFP2 fusion protein, we cloned the sequence of murine CD3ζ in frame with PS-CFP2 into the retroviral expression vector pIB2.
Alexa Fluor 647 (AF647)-conjugated TCRβ-specific antibody (clone H57-597); average degree of labeling of 7.4) was purchased from Biolegend (CatNo. 109218; LotNo. B206104). CD3ε-specific antibody (clone KT3) was from AbD Serotec/Bio-Rad Technologies (CatNo. MA1-80783; LotNo. 1603) and was conjugated to AF647 via NHS-ester chemistry following the supplier’s instructions. After removal of unreacted dye using Zeba desalting columns (Thermo Fisher Scientific) an average degree of labeling of 3.8 was determined.
For expression of the CD3ζ-PS-CFP2 fusion protein, we cloned the sequence of murine CD3ζ in frame with PS-CFP2 into the retroviral expression vector pIB2.
Alexa Fluor 647 (AF647)-conjugated TCRβ-specific antibody (clone H57-597); average degree of labeling of 7.4) was purchased from Biolegend (CatNo. 109218; LotNo. B206104). CD3ε-specific antibody (clone KT3) was from AbD Serotec/Bio-Rad Technologies (CatNo. MA1-80783; LotNo. 1603) and was conjugated to AF647 via NHS-ester chemistry following the supplier’s instructions. After removal of unreacted dye using Zeba desalting columns (Thermo Fisher Scientific) an average degree of labeling of 3.8 was determined.