Intercept buffer

The MC38 cell line was initially a kind gift from Mark Smyth, QIMR Berghofer Medical Research Institute) and has been used by our group previously^{25 , 26 (link)}. Cells were cultured in IDMD media(Thermo Fisher, Cat. # 12440046) supplemented with 10% fetal bovine serum (Thermo Fisher, Cat. # A3840001) and 1% penicillin/streptomycin (Thermo Fisher, Cat. # 15070063). Cells were harvested at ~80% confluency for subcutaneous injection into the flanks of female and male C57BL/6 mice (Jackson Laboratories) (n= 5). For mouse splenocytes, spleens were harvested from C57BL/6 mice right after euthanasia (n=3). Spleens were cut into ~1 mm pieces by clean surgical scissors in RPMI 1640 media (Thermo Fisher, Cat. # 11875101) and ground against a 70 μm cell strainer using the back end of a 10 mL syringe plunger. The cell strainer was washed with 10 mL RPMI1640 media to collect cells from homogenized tissue. Collected cells were incubated in ACK lysis buffer (Lonza, Cat. # 10–548E) for 3 minutes on ice for blood cell removal and washed in PBS. Isolated splenocytes were fixed immediately in 4% paraformaldehyde for 10 minutes, washed in PBS, and stored in Intercept buffer (Li-cor Biosciences, Cat. # 927–70001).

For protein extraction, RIPA buffer containing 1:100 protease and phosphatase inhibitor was added to the cells, then lysate was incubated at 4°C for 30 mins prior to centrifugation for 15 mins. Supernatant was harvested and stored at −80°C. Protein concentration was quantified using BCA assay. Blotting: 30 ug of protein was diluted 1:1 in 50:1 Laemmli buffer: B-mercaptoethanol and boiled at 95°C for 5 minutes. 8% gels were run at 130V for 90 mins. Protein was transferred to an Immobilon-FL membrane in Tris-Glycine buffer. Membranes were blocked in Intercept buffer (Licor) for 1 hour, then incubated in primary antibody diluted in Intercept buffer overnight at 4°C and secondary antibody (1:15000) for 1 hour. Membranes were imaged on a Licor instrument and quantified. Antibodies and concentrations are listed in Supplementary Table 3.