Antimouse mhc 2 antibody

FACS-sorted LECs from Sp19F-infected mice were co-cultured with OT-II CD4⁺ T cells (1:10 ratio) with 25μg/mL of OVA_323-339 peptide (Anaspec) in media containing 25% DMEM (Gibco), 25% F-12 (Gibco), 50% RPMI 1640 (Gibco) supplemented with 10% Heat inactivated FBS, Penicillin/Streptomycin (100 U/mL, Gibco), 1X Non-essential amino acids (Gibco), 2 mM L-Glutamine (Glutamax, Gibco), 1 mM Na-pyruvate (Gibco), 10 mM HEPES (Gibco), 0.2 μM beta-mercaptoethanol, 10 μg/mL Insulin, 50 mM Hydrocortisone, 100 μg/mL Transferrin, 1 mM Na-selenite, and 1 mM beta-estradiol for 8 h at 37 °C 5% CO₂ incubator. For assays with C57BL/6J LECs, EasySep™ Mouse CD4⁺ T Cell negative selection and Isolation Kit (StemCell Technologies, Cat# 19852) was used to enrich OT-II CD4⁺ T cells from spleens of OT-II TCR transgenic mice and 10 μg/mL of antimouse MHC-II antibody (Biolegend) was used for MHC-II blockade. For assays with MHC-II^ΔEpi LECs, EasySep™ Mouse CD4⁺ T Cell negative selection and Isolation Kit (StemCell Technologies, Cat# 19852) was used to enrich OT-II CD4⁺ T cells from spleens of OT-II TCR transgenic mice. This was followed by MHC-II blockade of the enriched CD4⁺ T cell suspension to block any excess MHC-II expressing splenocytes before rigorous washing and coculture with LECs for 8 h.

As previously described [19 (link)], each group's DCs and T cells that were going to be examined in the coculture system were first collected, then washed twice with PBS after being digested with 0.25 percent trypsin. After this, the cells were tested. Following centrifugation, the supernatant was discarded, and the cells were resuspended in Eppendorf (EP) tubes at a concentration of 2 105 cells per 100 μL. Then, l μL of primary antibodies was added to the EP tube, including antimouse CD133 antibody (BioLegend, USA), antimouse CD11c antibody (BioLegend, USA), antimouse CD80 antibody (BioLegend, USA), antimouse CD86 antibody (BioLegend, USA), antimouse MHC-II antibody (BioLegend, USA), antimouse CD4 antibody (BioLegend, USA), antimouse CD8 antibody (BioLegend, USA), and antimouse FOXp antibody (BioLegend, USA). Following a thirty minute incubation period in the dark at a temperature of four degrees Celsius, the cells were resuspended in 0.2 milliliters of PBS before being washed twice with PBS. The phenotypic changes in DCs and T cells were identified with the help of flow cytometry (a BD FACSCalibur), and the analysis of the experiment's results was performed with the FlowJo software tool (FlowJo™ v10.7, http://www.flowjo.com).