Tumor tissue was digested in the RPMI 1640 medium (Gibco) containing DNAse (60 µg/ml), hyaluronidase (0.2 µl/ml), Collagenase IA and IV (0.2 mg/ml each) for 30 min at 37 °C. Required enzymes were obtained from Sigma. Cell solutions were subsequently washed with RPMI and filtered using a 40 -µm cell strainer to obtain single-cell preparations.
Splenocytes were released from spleens by passing through a 40 -µm cell strainer and washed with RPMI 1640 (Gibco). Erythrocytes were lysed by adding 1x EBC lysis buffer (BioLegend) for 5 min at 4 °C, and cells were subsequently washed with RPMI 1640. Cells were resuspended in the RPMI 1640 medium supplemented with 2% FCS, 100 U/mL streptomycin, 100 mg/mL penicillin, 1% MEM with nonessential amino acids (×100 solution, Gibco) ß-mercaptoethanol (50 µM, Sigma), and sodium pyruvate (1 mM, Gibco), and kept at 37 °C and 5% CO2 overnight during peptide stimulation.
Erythrocytes were removed from blood samples by adding ×1 EBC lysis buffer (BioLegend) and incubated for 3 min at 4 °C. Cells were subsequently washed with RPMI 1640 (Gibco).
Splenocytes were released from spleens by passing through a 40 -µm cell strainer and washed with RPMI 1640 (Gibco). Erythrocytes were lysed by adding 1x EBC lysis buffer (BioLegend) for 5 min at 4 °C, and cells were subsequently washed with RPMI 1640. Cells were resuspended in the RPMI 1640 medium supplemented with 2% FCS, 100 U/mL streptomycin, 100 mg/mL penicillin, 1% MEM with nonessential amino acids (×100 solution, Gibco) ß-mercaptoethanol (50 µM, Sigma), and sodium pyruvate (1 mM, Gibco), and kept at 37 °C and 5% CO2 overnight during peptide stimulation.
Erythrocytes were removed from blood samples by adding ×1 EBC lysis buffer (BioLegend) and incubated for 3 min at 4 °C. Cells were subsequently washed with RPMI 1640 (Gibco).