Easysep cd8 t cell negative selection kit

In vitro stimulation T cell assay by OVA_257-264, GA-OVA, CBP-12-OVA or PBS was performed as described previously 23 (link). FL-DCs were incubated with 10 μg/mL OVA_257-264, GA-OVA, CBP-12-OVA or PBS for 1.5 h at 4 °C, and then the peptide-loaded FL-DCs were washed three times. Meanwhile, CD8⁺ T cells were isolated using EasySep^TM CD8⁺ T cell negative selection kit (STEMCELL, Vancouver, BC, Canada) from OT-1 mice. After labeling CD8⁺ T cells with 2 μΜ CFSE (eBioscience, San Diego, CA, US), 1 × 10⁵ CD8⁺ T cells per well were plated into round-bottom 96-well plates. After 30 min, 1 × 10⁴ peptide-loaded FL-DCs per well were gently added to the plates and incubated with CD8⁺ T cells for 72 h. Proliferation of DC-primed CD8⁺ T cells was determined by CFSE dilution. The cells were incubated with anti-mouse-CD3-PerCP-eFlour710 (17A2) and anti-mouse-CD8-APC (53-6.7) or Rat IgG2a-APC kappa Isotype Control (eBR2a), CD8⁺ T cells proliferation were detected by a flow cytometry. Supernatant was assayed for IFN-γ by a Mouse IFN-gamma ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA).

In vitro peptide stimulation T cell assays were performed using a modification of a previously described protocol [32 (link)]. In brief, 10μg/mL peptides (peptide-OVA_257-264, OVA_257-264, WH or NS) were incubated with FL-DC Day12 for 1.5 h at 4°C, by which the end time the peptide-loaded FL-DCs were washed three times. Meanwhile, OT-I CD8⁺ T cells were isolated using EasySep^TM CD8⁺ T cell negative selection kit (Stemcell Technologies). Subsequently, the CD8⁺ T cells were seeded at 10⁵ cells per well at round-bottom 96-well plates for 30 min. The peptide-loaded FL-DCs were then gently added at 10⁴ cells per well to the final volume of 200μL. After incubation for 72 h, Supernatants were assayed for IFN-γ by ELISA. Perforin and granzyme mRNA expression were tested by qRT-PCR. The perforin forward and reverse primers are 5′-AGCACAAGTTCGTGCCA GG-3′ and 5′-GCGTCTCTCATTAGGGAGTTTT T-3′, respectively. The granzyme B forward and reverse primers are 5′-CCACTCTCGACCCTACATGG-3′ and 5′- GGCCCCCAAAGTGACATTTATT-3′, respectively. The β-actin forward and reverse primers are 5′-GTGGCATCCATGAAACT ACAT-3′ and 5′- GGCATAGAGGTCTTTACGG-3′, respectively. For T cell proliferation assay, the CD8⁺ T cells were stained with 2μM CFSE. Three days later, CD8⁺ T cell proliferation was determined by CFSE dilution.

Single-cell suspension of splenocytes were prepared to isolate DCs, macrophages, eosinophils, CD4⁺ and CD8⁺ T cells. DCs were isolated by DC isolation micro-bread kit (StemCell) in line with manufacturer’s instructions. DCs with a purity of >90% were used for experiments. For macrophages isolation, splenocytes were plated in DMEM supplemented with 10% FBS at 37 °C in a humidified 5% CO₂ incubator for 1 h, then the supernatant fraction was poured off and the adherent splenocyte fraction was processed for CD68⁺ macrophages isolation by using EasySepTM PE positive selection kit (StemCell) in line with manufacturer’s instructions. Macrophages with a purity of >90% were used for experiments. Siglec-F⁺ eosinophils were isolated by using EasySepTM PE positive selection kit (StemCell) in line with manufacturer’s instructions. Eosinophils with a purity of >90% were used for experiments. CD4⁺ and CD8⁺ T cells were isolated by Easysep^TM CD4⁺ T cell negative selection kit and Easysep^TM CD8⁺ T cell negative selection kit (StemCell), respectively. CD4⁺ and CD8⁺ T cells with a purity of >90% were used for experiments.