Polystyrene round bottom tube

A Tumor Dissociation Kit, gentleMACS C Tubes, gentleMACS Disssociator, and MACSmix Tube Rotator were obtained from Miltenyi Biotec (Auburn, CA, USA). A Mouse CD3⁺ T Cell Isolation Kit, Mouse CD8⁺ T Cell Isolation Kit, polystyrene round-bottom tube, and Magnet for column-free immunomagnetic separation were obtained from STEMCELL Technologies (Vancouver, BC, Canada). Dynabeads Mouse T-Activator CD3/CD28 was purchased from Life Technologies (Carlsbad, CA, USA). A Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Oxa was purchased from TargetMol (Wellesley, MA, USA). Mouse IL-2 ELISA was purchased from BD Biosciences (San Diego, CA, USA). Mouse GrB ELISA was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD8 antibody and anti-GrB antibody were obtained from Cell Signaling Technology (Danvers, MA, USA).

B cells (CD19+, CD19+ CD138+ and CD138+) including plasma cells were isolated by immunomagnetic negative selection from total lymphocyte suspension by using EasySep Mouse B-Cell Isolation Kit (Stem Cell Technologies, catalog no. 19854). In brief, lymphocyte suspension was prepared (1 × 10⁸ cells/ml in 0.25–2 ml PBS, 0.5% BSA) in 5-ml polystyrene round-bottom tube (12 × 75 mm) (Stem Cell Technologies, catalog no. 38007) and rat serum was added (50 µl/ml of cell suspension). Isolation cocktail containing biotinylated antibodies recognizing specific cell surface markers for non-B cells was added to the sample (50 µl/ml of cell sample) and mixed and incubated for 5 min at RT. Magnetic beads (EasySep Streptavidin RapidSpheres 50001) were added to the sample (50 µl/ml of cell suspension), mixed and incubated for 3 min at RT. The volume of the sample was made to 2.5 ml by adding PBS, 0.5% BSA. Non-B cells labeled with biotinylated antibodies and streptavidin-coated magnetic particles were discarded by magnetic separation (EasySep Magnet, Stem Cell Technologies, catalog no. 18000) and total B cells were isolated in solution. Cell number was determined using Vi-cell XR cell counter (Beckman Coulter).