Easysep mouse naive cd4 t cell isolation kit

2 × 10⁴/well of DCs were plated in 96 well round-bottom plates in RPMI (Thermo Scientific) supplemented with 10% FBS, 5 ng/ml GM-CSF and stimulated with either C. albicans or polystyrene beads overnight. The next day, DCs were pulsed with 500 nM Ova 323–339 peptide (Anaspec) for 2 h and then co-cultured with 2 × 10⁵ of naïve Rag1^−/− or Rag2^−/− OT-II TCR transgenic CD4⁺ T cells for 3 days. Naïve CD4⁺ T cells were isolated from spleens and axillary, brachial, inguinal, and mesenteric lymph nodes from Rag^−/− OT-II TCR transgenic mice and purified by using an EasySep mouse naive CD4⁺ T cell isolation kit (Stem Cell Technologies). For co-cultures with wild-type naïve CD4⁺ T cells, 1 ×10⁶ DCs were plated in non-tissue culture treated 12-well-plates and stimulated with either yeast of hyphal form of C. albicans overnight. The next day, 2 ×10⁴ of DCs were transferred to each well of 96 well round-bottom plates and pulsed with 500 nM Ova 323–339 peptide for 2 h and then co-cultured with 2 ×10⁵ of wild-type naïve CD4⁺ T cells.

Naive CD4⁺ T cells were purified from WT and RGC-32^−/− spleen cells using an EasySep mouse naive CD4 T cell isolation kit (Stemcell Technologies). Cells were then cultured under Treg differentiating conditions for 72 h. Treg differentiation was tested by induction of Foxp3 by FACS analysis. CD4⁺CD25⁺ Tregs were purified using a mouse Treg isolation kit II (Stemcell Technologies). For the suppression assay, 5 × 10⁴ purified CD4⁺CD25⁺ Tregs were cultured in triplicate with 5×10⁴ or 2.5 ×10⁴ purified CD4⁺CD25² T effectors (T responder) and 1 µg/ml anti-CD3 mAb in a 96-well round-bottom plate. T cell–depleted splenocytes from C57BL/6 mice (4×10⁵) irradiated with 3000 Gy were used as APCs. Proliferation of the T responder cells was determined by [³H]thymidine incorporation. Percentage suppression of T responder proliferation was calculated using the formula: 100 × [(cpm of T responder cells alone 2 cpm of T responder cells cocultured with Tregs)/cpm of T responder cells alone].

For human samples, naive CD4 T cells were isolated from previously frozen PBMCs using the EasySep^™ Human Naive T cell isolation kit II (STEMCELL Technologies, Inc., Vancouver, Canada) according to the manufacturer’s instructions. Naive CD4 T cells were then stimulated with Dynabeads^™ Human T-Activator CD3/CD28 (ThermoFisher Scientific) according to the recommended concentration for 2 to 3 days. Recombinant human IL-2 (Peprotech) was added at a final concentration of 100 IU/ml. For mouse samples, naive CD4 T cells were isolated from spleens and lymph nodes of standard laboratory diet- and WD-fed ApoE^−/− mice using the EasySep^™ Mouse Naive CD4 T cell isolation kit (STEMCELL Technologies, Inc.). Naive CD4 T cells were stimulated in αCD3-coated plates (2 μg/ml) and soluble αCD28 (1 μg/ml) was added to these wells for 2 days. All cells were cultured in RPMI with 10% FCS, L-glutamine, penicillin and streptomycin, and β-mercaptoethanol (for mouse cells). For the proliferation assays, isolated naive CD4 T cells were labeled with CTV (ThermoFisher) according to the manufacturer’s instructions, prior to stimulation. The percentage of cells divided was calculated using FlowJo v. 10.0.08 (Tree Star, Ashland, OR).

Spleen cells were excised from mice and single-cell suspensions of splenocytes were prepared in RPMI 1640 by teasing the organ through a sterile nylon mesh. Naive CD4⁺ T cells were purified using an EasySep mouse naive CD4⁺ T cell isolation kit (Stemcell Technologies, Vancouver, BC, Canada) and cultured with plate-bound anti-CD3 (5 µg/ml; Bio X Cell), anti-CD28 (5 µg/ml; Bio X Cell), and cytokines used alone, including IL-12 (R&D Systems), IFN-γ (R&D Systems), IL-1 (R&D Systems), IL-4 (R&D Systems), IL-6 (Cell Signaling Technology), IL-23 (eBioscience), and TGF-β (Invitrogen), or in combination with Abs (Bio X Cell) for Th17-promoting (2.5 ng/ml TGF-β, 20 ng/ml IL-6, 10 µg/ml anti–IFN-γ, 10 µg/ml anti–IL-4, 10 µg/ml anti–IL-2), Th1-promoting (10 ng/ml IL-12, 5 µg/ml anti–IL-4), Th2-promoting (10 ng/ml IL-4, 5 µg/ml anti–IFN-γ), or regulatory T cell (Treg)–promoting (5 ng/ml TGF-β, 5 µg/ml anti–IFN-γ, 5 µg/ml anti–IL-4) conditions. In some experiments, naive CD4⁺ T cells were cultured in Th17-promoting conditions and IL-21 (20 ng/ml; eBioscience) or IL-23 (50 ng/ml) was added to cultures after 24 h. Cells were harvested after 48 h for real-time PCR analysis and after 72 h for cytokine expression by flow cytometry as previously described (23 (link)).