Cd4 t cell macs separation kit

CD11c^highMHCII^high DCs were purified from lymph nodes of C57BL/6 and Batf3^−/− mice as described above. For the isolation of OVA-specific naive T cells, spleens were obtained from C57BL/6 Rag1^−/− OT-II Tg mice. Naive CD4⁺ T cells were enriched using CD4⁺ T cell MACS separation kit (Miltenyi Biotech) and subsequently FACSsorted based on the expression of CD4, CD45RB and CD25 to obtain >99% pure population. The purified naive OVA-specific CD4⁺ T cells were labeled with CFSE (Invitrogen) at a final concentration of 2 μM. For the in vitro stimulation assays, dendritic cells were cultured with 2 mg/ml OVA protein for 1 hours at 37 °C. Subsequently, cells were washed extensively with sterile PBS after which the CFSE-labeled T cells were added at a 1:10 ratio in the presence or absence of 2 ng/ml hTGFβ (R&D Systems Inc). At 72 hours, cells were analyzed for division and Foxp3 expression.

CD4⁺ T cells were purified from inguinal lymph nodes (draining immunization site) of Batf3^−/− mice after induction of a DTH reaction using the CD4⁺ T cell MACS separation kit (Miltenyi Biotech). For DC isolation, spleens from untreated C57BL/6 wild-type mice were isolated and digested with Liberase TL (Roche, Indianapolis, IN, USA) in the presence of DNAse I (Roche) at 37 °C. CD11c⁺ cells were purified from the digested cell suspensions using a CD11c⁺ DC MACS separation kit (Miltenyi Biotech). For in vitro restimulation, 1×10⁴ CD11c⁺ cells were pulsed with 0.5 mg/ml OVA protein and cocultured with 1×10⁵ CD4⁺ T cells for 48 hours. Alternatively, 5×10⁵ cells from the inguinal lymph nodes of MLN-resected mice were restimulated with OVA protein. Subsequently, protein levels of IFNγ were measured in culture supernatant by mouse ELISA set kit (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. The sensitivity of the assay was 4 pg/ml.