Glutamax

Primary mouse calvarial osteoblasts were isolated following a protocol previously described [26 (link)]. Cells were obtained by sequential enzymatic digestion using Collagenase type 2 for 10 minutes at 37^o C. Collected cells were maintained in α-MEM supplemented with antibiotic/antimycotic (ThermoFisher Scientific), Glutamax and FBS 10% (GE Healthcare Life sciences HyClone laboratories). For osteoblast differentiation, confluent cells were cultured with supplemented α-MEM or supplemented α-MEM containing 5 mM CaCl₂, and allowed to differentiate for 0, 3, 7 and 10 days. SB431542, a selective TGFB type I receptor inhibitor (Tocris), was solubilized using DMSO at a 10 mM stock concentration, and a final concentration of 10 μM. AG-1296, a selective PDGF receptor inhibitor (Abcam), was used at a final 10 μM concentration.

Wild type Jurkat cells (TIB-152), K562 cells (CCL-243), HEK293T cells (CRL-3216), and T2 (CRL-1992) cells were obtained from the ATCC. J76 cells, a TCR negative Jurkat cell clone, are a generous gift from Mark Davis Lab from Stanford. Jurkat, J76, K562 cells were maintained in RPMI 1640 media (Cytiva, SH30096.FS) supplemented with 10% FBS (Sigma, F0926) and 10uM Glutamax (Gibco, 35050061). T2 cells were maintained in DEME media (Cytiva, SH30243.FS) supplemented with 10% FBS and 10uM Glutamax. To established HLA-A*03:01 monoalleic antigen presenting cell line, WT K562 were electroporated with a sleeping beauty transposase plasmid (VectorBuilder pRP[Exp]-CMV>T7/SB100X) and a sleeping beauty transposon plasmid (VectorBuilder sleeping beauty backbone, VB230803–1359aaa) co-expressing HLA-A*03:01 gene, B2M gene, and mNeonGreen gene as described previously^{63 (link)}. The plasmids were constructed with VectorBuilder service (vector ID: VB900088–2243bzq). The electroporation was performed with Lonza 4D unit (Protocol CL-120) and 2ug total plasmid DNA per 1e6 cells. HLA-A*03:01 positive cells were sorted with Sony cell sorter SH800S based on the surface HLA level and GFP level (WT K562 cells have none to low HLA levels).