Ls004188

Tissue was dissociated using the following steps. Fat layers were removed, washed, and opened longitudinally. Tissues were then minced and dissociated in a cocktail solution of 12 mg collagenase IV (LS004188; Worthington), 180 U DNase (D5025; Sigma) and 1.2 mg hyaluronidase (H3506; Sigma) in 20 ml complete RPMI-1640 media with constant stirring for 25 min at 37 °C. Single cell suspensions were then filtered, and supernatants were washed in PBS-2% FBS. Tissues were digested twice. A percoll (P1644; Sigma) gradient was then performed to remove platelets and debris by layering the 44 % percoll cell suspension over 67 % percoll and centrifuging at 400 g for 20 min at 4 °C without brake. The mononuclear cell layer was collected and washed in PBS-2% FBS buffer.

Germ cell sorting was as described (Gainetdinov et al., 2018 (link); Yu et al., 2022 (link)). Briefly, we de-capsulated the testis specimens and incubated with 1× Gey′s Balanced Salt Solution (GBSS, Sigma, G9779) containing 0.4 mg/ml collagenase type 4 (Worthington; LS004188) at 33°C for 15 min. Thereafter, we washed the seminiferous tubules twice with 1× GBSS, and incubated them with 1× GBSS containing 0.5 mg/ml trypsin and 1 μg/ml DNase I at 33°C for 15 min. Next, the seminiferous tubules were gently pipetted up and down for 3 min through a Pasteur pipette to homogenize at 4°C on. Trypsin was inactivated with fetal bovine serum (FBS; f.c. 7.5% [v/v]), and the cell suspension was passed through a pre-wetted 70 μm cell strainer. Cells were recovered by centrifugation at 300 × g at 4°C for 10 min, resuspended in 1× GBSS containing 5% (v/v) FBS, 1 μg/ml DNase I, and 5 μg/ml Hoechst 33342 (Thermo Fisher, 62249), and incubated at 33°C for 45 min rotating at 150 rpm. Finally, we added propidium iodide (0.2 μg/ml, f.c.; Thermo Fisher, P3566) directly to the cells which were further filtered through a pre-wetted 40 μm cell strainer. Cell sorting (FACS Core, University of Massachusetts Medical School) was performed as described (Bastos et al., 2005 (link); Yu et al., 2022 (link)).

Single-cell suspensions were prepared from dissected tumors and spleens. Spleens were mechanically disrupted using a plunger end of a 5 mL syringe and resuspended in 1% FBS/PBS after passing through a 70-μm cell strainer (Falcon). Red blood cells were depleted from total splenocytes using 1x RBC Lysis Solution (eBioscience, 00–4333-57). For isolation of tumor-infiltrating lymphocytes, tumor tissue was minced into 1 to 2 mm pieces and digested with collagenase IV (1.25 mg/mL; #LS004188, Worthington), 0.1% trypsin inhibitor from soybean (# T9128, Sigma), hyaluronidase (1 mg/mL; # LS 002592, Worthington), and DNase I (100 mg/mL; # LS002007, Worthington) in complete DMEM for 30 minutes at 37°C. Cell suspensions were passed through a 70-μm cell strainer (Falcon) and resuspended in RPMI media (Gibco). Lymphocytes were isolated from processed tumor tissues by OptiPrep (Sigma) density gradient centrifugation. For experiments requiring ex vivo stimulation and intracellular cytokine staining, regulatory B cells (CD19⁺CD1d^HiCD5⁺CD21^Hi) and conventional B cells (CD19⁺CD1d^LoCD5^-CD21^Lo) were sorted from single cell splenic suspensions using the BD FACS Aria III cell sorter according to manufacturer’s instructions and UNC flow cytometry core guidelines.