Do11.10 t cell receptor transgenic mice

DO11.10 T cell receptor transgenic mice (Jackson Laboratories) were bred in the POSTECH Biotech Center (PBC). All experiments involving mice, including euthanasia of mice and cell isolation, were performed in accordance with guidelines and regulations approved by the Institutional Animal Care and Use Committee at PBC. DO11.10 T cells were harvested from spleens and lymph nodes of DO11.10 mice and stimulated with 1 μg/ml of OVA323-339 peptide (ISQAVHAAHAEINEAGR, Peptron, Korea). DO11.10 T cells were grown in R10 media, RPMI 1640 media (Invitrogen) supplemented with 10% FBS (Gibco), 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen), and 0.1% beta-mercaptoethanol (Sigma). On day 2 of stimulation, 1 U/ml of IL-2 was added and cells on day 5 were used for experiments.

DO11.10 T cell receptor transgenic mice (Jackson Laboratories) bred in the POSTECH Biotech Center (PBC) were used to generate DO11.10 T cell blasts. All the T cell assay including mice treatments were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at PBC. DO11.10 blasts were obtained by stimulating cells in the spleen and lymph nodes of DO11.10 transgenic mice with 1 μg/ml of OVA323–339 peptide (ISQAVHAAHAEINEAGR, Peptron, Inc. Korea). Cells were cultured in RPMI 1640 (Invitrogen) containing 10% of FBS, 1% of penicillin-streptomycin and 0.1% of beta-mercaptoethanol (Sigma). 1–2 U/ml of IL-2 was added on the 2^nd day of stimulation and cells were used for experiments on the 5^th days. To inhibit arp2/3 complex, myosin light chain kinase (MLCK), ROCK and non-muscle myosin II, CK636 (100 μM, Sigma), ML7 (30 μM, Sigma), Y-27632 (50 μM, Sigma) and blebbistatin (50 μM, Sigma) were incubated with cells for 1 h, respectively. For arp2/3 complex inhibition, CK666, which is a more potent derivative of CK636^{35 (link)}, can be used, instead.