Live dead blue cell stain

Single-cell suspensions were obtained from tumor, spleen, peripheral blood, and cell culture. All tumors were minced by scissors and razor blade in 60 mm dishes with 5 mL of media (RPMI + PS, FBS-free) and then digested with 250 μL/5 mL media Liberase for 30 min at 37 °C. A quantity of 100 μL of 100 mM EDTA was added to stop 1 mL of enzymatic digestion (500 μL for 5 mL). Digested tissue was pipetted up and down 30 times using a glass Pasteur pipette and then passed through a 100-μm nylon filter to acquire single-cell suspensions. Spleens and peripheral blood were treated with 5 mL of ACK lysis buffer, followed rapidly by the addition of complete media (RPMI, 5% FBS, P/S). Cells were resuspended in flow cytometry staining buffer. All cells were stained with Live Dead Blue Cell Stain (ThermoFisher, Cat. #L23105) and stained at recommended dilution for 30 min with conjugated antibodies. Cell trace violet (CTV, ThermoFisher, Cat. #C34557) was used to label splenocytes in experiments involving CD8⁺ T cell proliferation.

3D-O matrices were enzymatically digested with collagenase (20 mg/ml for 2–3 h at 37°C) on day 4. BCa cells were isolated and identified by gating cells with a high DiO signal (excitation, 488 nm; emission, 530/30 nm). Antibodies used to evaluate hypoxic status and surface marker expression were AlexaFluor 647 conjugated anti-HIF-1 α (359706, BioLegend, CA), APC-Cy7 conjugated anti-CD8 (344714, BioLegend, CA), PE-conjugated anti-PD-L1 (393608, BioLegend, CA), PE-conjugated anti-MUC-1 (355608, BioLegend, CA), and PerCP-Cy5 conjugated anti-CD73 (344014, BioLegend, CA). Cell viability was evaluated by using a Sytox Blue live-dead fluorescent dye (S34857, Invitrogen, CA) possessing excitation, 358 nm; emission, 461 nm or Live/Dead Blue cell stain (L34962, Thermo Fischer Scientific, MA). For all analyses, a minimum of 5,000 events were acquired using BD FACS Fortessa and FACSDiva v6.1.2 software or BD FACS Accuri and BS Accuri C6 software (BD Biosciences), respectively. The BCa cell counts were always normalized to a predetermined number of counting beads (424902, BioLegend, CA), and mean fluorescence intensity (MFI) ratios for each of the targets as mentioned above studied were assessed with respect to the corresponding isotype in the BCa-DiO⁺ cells. The data was analyzed using FlowJo program v10 (Ashland, OR).

For the characterization of the innate and the GC B cell responses, cell suspensions prepared from spleen, or pairs of draining inguinal lymph nodes from each mouse, were stained with blue live/dead cell stain (Invitrogen) for 20 min at room temperature and then incubated with Fc block (BD Biosciences) in PBS plus 1% fetal bovine serum (HyClone, Thermo Scientific) for 10 min at 4°C. About 10⁷ splenocytes or 2 × 10⁶ to 3 × 10⁶ lymph node cells were stained for 30 min at 4°C with the following mAbs: anti-CD19 APC-H7 (BD Biosciences), anti-CD3 BV785 (BioLegend), anti-CD38 peridininchlorophyll- protein complex (PerCP)–C5.5 (BD Biosciences), and anti-CD95 BV510 (BD Horizon). Cells were analyzed on a FACS Fortessa (BD Biosciences), and data analyses were performed using FlowJo software v9.6 (Tree Star).