Dynal mouse cd4 cell negative isolation kit

CD4⁺ T lymphocytes from B6.Cg-Tg(TcraTcrb)425Cbn/J transgenic mice (known as OT-II mice), which are specific for the 323–339 peptide of ovalbumin (OVA), were purified from splenic and lymphatic nodes cells using the Dynal Mouse CD4 Cell Negative Isolation Kit (Invitrogen) and stained with 5 μM CFDA-SE (Thermo Scientific). BMDCs were seeded into 96-well round-bottomed plates (40 × 10³ per well), in the presence or absence of 1 μg pLL. After letting the BMDC settle for one hour, 200 × 10³ labelled T cells were added, in the presence or absence of 0.1 μg/mL OVA peptide. After three days of incubation, the co-culture was placed on ice and stained for flow cytometry.

Mesenteric lymph nodes (MLNs) were obtained from healthy SPF 10-week-old C57/BL6 male mice (SLAC Inc., Shanghai, China), then washed once and collected in PBS (Hyclone). CD4+ cells were separated from total cells using the Dynal® Mouse CD4 Cell Negative Isolation kit (Invitrogen, CA, USA). The CD4+ cells were placed in 96-cell tissue culture plates at a density of 1 × 10⁶ cells/well. CD4+ cells were co-cultured with 10% (v/v) BCS, or 10% unfermented synthetic medium, or 10% PBS (control) in DMEM with 5 ng/ml human TGF-β (R&D systems) and 10 ng/ml recombinant mouse IL-2 (R&D systems) for 72 h. The medium was changed every 36 h. Cells were collected by centrifugation at 1000 × g for 3 min (4°C), washed once, and resuspended in magnetic-activated cell sorting (MACS) buffer (PBS + 0.5 % BSA + 2 mM EDTA, pH = 7.2). The number of Foxp3+ Treg cells was assessed as described in Flow Cytometry.