Mouse rbc lysis buffer

Mice were implanted with 005 GSCs (1.0 × 10⁵ cells/mouse) and treated with intratumoral injection of ICOVIR17 (day 9: 1.6 × 10⁷ PFU/mouse, day 14: 1.0 × 10⁷ PFU/mouse) or PBS on days 9 and 14. Anti-mouse PD-1 antibody or isotype rat IgG (as control)(12.5 mg/kg) was injected ip on days 10, 13, 16, 19. Mice were euthanized on day 21. Tumor tissues or spleens were harvested from mice and mashed through a 100 μm strainer. For splenocytes, red blood cells were lysed using Mouse RBC lysis buffer (Boston BioProducts, IBB-198). Live/dead cell discrimination was performed using Zombie UV™ Fixable viability kit (BioLegend, 423108). Cells were incubated with FcR blocking reagent (Milteneyi Biotec, 130-092-575) followed by cell surface staining with fluorochrome-conjugated anti-mouse antibodies. Staining of intracellular FOXP3 was done after fixation and permeabilization by FOXP3 Fix/Perm buffer set (BioLegend, 421403), while other intracellular staining was done using BD Cytofix/Cytoperm™ Fixation/Permeabilization kit (BD, 554714). Samples were run in an LSR II flow cytometer (BD) and data was analyzed using FlowJo software (v.10.1, Tree Star).
Antibodies are listed in Supplementary Table S1.

Mice were implanted with 005 GSCs (1.0 × 10⁵ cells/mouse), and treated with intratumoral injection of ICOVIR17 (1.6 × 10⁷ PFU/mouse) or PBS on days 9 and 14. Anti-mouse PD-1 antibody or isotype rat IgG (both 12.5 mg/kg) were injected ip on days 10, 13, 16, and 19. On day 21, spleens were collected, passed through a 70 μm strainer, and incubated with mouse RBC lysis buffer (Boston BioProducts, IBB-198) to remove erythrocytes. Splenocytes were cultured in RPMI with 10% FCS and IL-2 (100 U/ml) for 48 h at 37°C and 5% CO₂, and seeded in 96-well plates pre-seeded with 005-Fluc or B16.F10-Fluc cells for different E: T ratios. After 24 h-co-culture, cell viability of tumor cells was measured by luciferase assay.

C57BL/6 mice or BLT mice were intrathecally implanted with UV2-GFl (5 × 10⁴ cells per mouse) or M12-GFl (5 × 10⁴ cells per mouse), respectively, and treated with intrathecal injection of SC-based therapy on day 5. On day 12 or 15, mice were euthanized, and tumors were collected. Tumor tissues or spleens were harvested from mice and mashed through a 100-μm strainer. For splenocytes, RBCs were lysed using mouse RBC lysis buffer (Boston BioProducts, IBB-198). Live/dead cell discrimination was performed using the Zombie UV Fixable Viability Kit (BioLegend). Cells were incubated with FcR blocking reagent (Miltenyi Biotec) or Human TruStain FcX (Fc receptor blocking solution) (BioLegend), followed by cell surface staining with fluorochrome-conjugated anti-mouse Abs or anti-human Abs. Stained cells were fixed with 4% paraformaldehyde before running them on BD Fortessa. FCS files were analyzed on FlowJo (version 10.3.1).