70 μm pore nylon cell strainers

After resection, tumors were kept in normal saline with antifungal and antibiotic agents. Normal tissue and fat were removed, and the tissues were rinsed with 1× Hanks' balanced salt solution (HBSS). Tumor tissue was minced with dissociation medium, which contained RPMI-1640 medium (Hyclone, South Logan, UT, USA) supplemented with 20% FBS and 1 mg/mL of type IV collagenase (Sigma-Aldrich, St Louis, MO, USA). Minced and suspended tumor cells were filtered through sterile 70-μm-pore nylon cell strainers (BD Falcon, Franklin Lakes, NJ, USA), rinsed with 50 mL 1× HBSS, and centrifuged at 1400 rpm for 5 min. Cells were resuspended with RPMI-1640 medium containing 10% fetal bovine serum (FBS; Hyclone) and 2% penicillin/streptomycin solution (Gibco, Grand Island, NY, USA). Cell viability was analyzed by the trypan blue dye exclusion method.

Naïve B cells were isolated from the spleen. Spleens were meshed through 70 μm pore nylon cell strainers (BD Falcon) in complete RPMI medium supplemented with 10% FBS and penicillin (50 U/ml) and streptomycin (50 μg/ml). Erythrocytes were lysed by incubating the cell suspension in erythrocyte lysis buffer (ACK Lysing Buffer, BioWhittaker) for 4 minutes at room temperature. After washing with cold complete RPMI, B cells were isolated by immunomagnetic depletion using anti-CD43 beads (Miltenyi Biotec). B cell purity was >95%.