CD8+ T cells were purified from spleens of OT-I mice by negative selection with magnetic beads (EasySep, Stemcell Technologies). After purification, cells were 97.7±0.5% CD8+ T cell and contained 0.11±0.04% CD11b+ CD11c- monocytes and 0.09±0.05% CD11b+ CD11c+ dendritic cells. In each well of a 24-well plate, 5x105 of the purified CD8+ T cells/ml were cultured in complete media (RPMI 1640, 10% FBS (Gibco), 1% 2mM L-glutamine (Life Technologies), 1% HEPES (Life Technologies), 1% 100nM Sodium Pyruvate (Life Technologies), 1% non-essential amino acides (Life Technologies), 100U/ml penicillin (Gibco) and 100μg/ml Streptomycin-sulfate (Gibco), 0.05mM Betamercaptoethanol (Sigma)) with IL-15 (5ng/ml, Peprotech, Cat 210–15) and IL-7 (5ng/ml, Peprotech, Cat 210–07) with or without 10ng/ml OVA(257–264) peptide (Anaspec Cat AS-60193).
For single peptide stimulation, cells were cultured in the presence of OVA(257–264) peptide for 48 hours. The peptide was then removed by washing the cells two times with complete media. For the remaining 3 days, the cells were cultured in the complete media with cytokines. For repeat peptide stimulation, 10ng/ml OVA(257–264) peptide was added daily for five days. The cells were washed also on day 2 to allow for comparable culture conditions. Unstimulated control cells were cultured in media with cytokines but without adding peptide. Cells from all three conditions were checked daily, and when the cells were confluent, they were split and cultured with fresh complete media containing cytokines. After day 5, some of the cell were washed two times with complete media and maintained in the media only with cytokines for another three days. In some experiments cell cultures were treated on day 2 with 20μM DNA methyltransferase (DNMT) inhibitor SGI-1027 (Tocris, Bio-techne) that targets DNA methyltransferases DNMT3B, DNMT3A and DNMT1.
On day five, cells were harvested and counted using an automated counting system (Countess, Life Technologies). Cells were stained with DAPI Viability dye (Beckman Coulter, Cat B30437) and Acridine Orange (Biotium, Cat 40039) to distinguish live and dead cells.
For single peptide stimulation, cells were cultured in the presence of OVA(257–264) peptide for 48 hours. The peptide was then removed by washing the cells two times with complete media. For the remaining 3 days, the cells were cultured in the complete media with cytokines. For repeat peptide stimulation, 10ng/ml OVA(257–264) peptide was added daily for five days. The cells were washed also on day 2 to allow for comparable culture conditions. Unstimulated control cells were cultured in media with cytokines but without adding peptide. Cells from all three conditions were checked daily, and when the cells were confluent, they were split and cultured with fresh complete media containing cytokines. After day 5, some of the cell were washed two times with complete media and maintained in the media only with cytokines for another three days. In some experiments cell cultures were treated on day 2 with 20μM DNA methyltransferase (DNMT) inhibitor SGI-1027 (Tocris, Bio-techne) that targets DNA methyltransferases DNMT3B, DNMT3A and DNMT1.
On day five, cells were harvested and counted using an automated counting system (Countess, Life Technologies). Cells were stained with DAPI Viability dye (Beckman Coulter, Cat B30437) and Acridine Orange (Biotium, Cat 40039) to distinguish live and dead cells.
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