Easysep cd4 t cell enrichment kit

Splenic CD4 T cells from OT-II and OT-II.Ncf1^m1J mice were purified by negative selection according to the manufacturer’s protocol using the EasySep CD4 T cell enrichment kit (STEMCELL Technologies). CD4 T cell purity was routinely assessed by flow cytometry and found to be greater than 90% (data not shown). T cells were stimulated with plate-bound anti-CD3ε (0.1μg/mL) and anti-CD28 (1μg/mL) (6 (link)). CD4 T cell responses were assayed via antigen recall assays, as described previously, in which splenocytes were stimulated with 1μM OVA_323-339 (3 (link)). To assess the ability of exogenous superoxide to rescue Th1 T cell responses, OT-II and OT-II.Ncf1^m1J splenocytes were treated with potassium superoxide as described (22 (link)). A titration response (0.5, 1, and 5μg/mL) with potassium superoxide was performed in preliminary studies with OT-II and OT-II.Ncf1^m1J splenocyte primary recall assays and 0.5μg/mL was chosen as the ideal concentration to restore Th1 cytokine responses without compromising cell viability. MTT assay for viability assessment and cell counting kit-8 (CCK-8) assay for proliferative capacity were performed according to manufacturer’s protocol (21 (link), 23 (link)).

FRC were sorted from naïve mice as described above and used in the following conditions: naïve (sorted on the same day); or stimulated with agonistic anti-CD40 mAb (10 μg/ml, clone FGK4.5) overnight. Naïve CD4⁺ T cells were isolated from spleen by EasySep CD4⁺ T cell enrichment kit (STEMCELL Technologies, Vancouver, BC, Canada), labeled with CFSE (5μM) (Molecular Probes/Life Technologies, Grand Island, NY) according to the manufacturer's protocol and added to the FRC. T cell culture conditions were as follows: 1) IL-2 only (20 ng/ml, R&D Systems, Minneapolis, MN); 2) IL-2 and soluble anti-CD3 mAb (eBioscience) (0.3 μg/ml); and 3) IL-2, anti-CD3 and soluble anti-CD28 (eBioscience) (0.3 μg/ml) mAbs. FRC and CD4⁺ T cells (5 × 10⁴) were cultured in 200μl in 96 well plates. 5 × 10⁴ CD4 depleted, 800 rad irradiated splenocytes were used as control antigen presenting cells (APC). CD4⁺ T cell proliferation by CSFE dilution was measured on day 3 by flow cytometry.

To assess CD4 T cell migration, BM-Mɸ were seeded and differentiated onto the lower chambers of a 24-well Corning transwell plate containing 8 μm pores. Splenic CD4 T cells from diabetogenic NOD.C6.BDC-6.9 mice were purified by negative selection according to the manufacturer’s protocol using the EasySep CD4 T cell enrichment kit (STEMCELL Technologies). CD4 T cell purity was routinely assessed by flow cytometry and found to be greater than 90% (data not shown). Following stimulation with p(I:C) in the presence or absence of (PVPON/TA) capsules for 24 hours, 2×10⁵ C6.BDC-6.9 CD4 T cells were added to the upper chamber. Following incubation at 37°C for 24 hours, non-adherent T cells were collected from the lower chambers and counted via trypan blue exclusion.