Mcd45.2 apc 104

The Jurkat E6.1 cells and the mouse thymoma cell line BW5147 were derived from in-house stocks and cultured as previously described [52 (link)]. All cell lines were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) (all from Sigma Aldrich, St. Louis, MO, USA). Cell lines were tested for mycoplasma contamination using a previously described reporter system [53 (link)]. The generation of the Jurkat NFκB-eGFP cell line and the T cell stimulator cell line (TCS) has been described previously [34 (link),35 (link)]. The T cell stimulator cell line expresses a membrane-bound human CD3 antibody single-chain fragment, which can activate T cells by engaging their CD3/TCR complex [35 (link)]. To assess the surface expression of the receptors, the following antibodies were purchased from Biolegend (San Diego, CA, USA): HVEM-PE (122), BTLA-APC (MIH26), CD14-APC (63D3), and mCD45.2-APC (104). Flow cytometry analysis was performed on a FACSCalibur™ flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). FlowJo software (version 10.6.1, Tree Star, Ashland, OR, USA) was used for flow cytometry data analysis.