Tissue digestion buffer

The LKR-13 K-RasG12D lung adenocarcinoma cell line was obtained from J. Kurie (MD Anderson). The reagents obtained, used and cells were cultured in RPMI 1640 as previously described [3 (link)]. Immunofluorescence (IF) Ab for perforin and PD-L1 were from CST, granzyme B conjugated to Alexa 647 and Pan-CK were from Abcam, CD4 and CD8 conjugated to Alexa 488 were from Novus Biological. IF buffers were from DAKO and blocking buffer was from Thermo Fisher. For in vivo experiments, anti-PD-1 (BE0146), anti-CD4 (L3T4) anti-CD8 (YTS169.4), and anti- CXCL9 (MIG-2F5.5) were from BioXCell as previously described [3 (link)]. Goat anti-mouse CXCL10 was given by Dr. Robert Strieter. Isotype control Ab was from Sigma. Tissue digestion buffer was from Miltenyi and used according to manufacturer’s instructions. T cell purification columns were from R&D. RNA isolation kit was from Qiagen.

The murine K-RasG12Dp53null lung adenocarcinoma was isolated from lung tumors of the K-RasG12Dp53null transgenic mice obtained from J. Kurie (MD Anderson Houston, TX, USA). The culture medium (CM) was RPMI 1640 supplemented with 10% fetal bovine serum (Gemini Bioproducts West Sacramento, CA, USA), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM glutamine (JRH Biosciences Lenex, MA, USA). Fluorescein isothiocyanate-, phycoerythrin-, allophycocyanin-, PerCP-, or APC-Cy7-conjugated anti-mouse Abs to CD4 (RM4-5) and CD8a (53-6.7) were from BD Biosciences or eBiosciences. For in vivo experiments, anti-PD-1 (BE0146), anti-CD4 (L3T4), and anti-CD8 (YTS169.4) were from BioXCell. Isotype control antibody (Ab) was from Sigma. The Abs used for T cell depletion were different for monitoring T cell levels. Bradford protein quantification dye was obtained from Sigma. Tissue digestion buffer was obtained from Miltenyi and used in Miltenyi tissue homogenizer according to the manufacturer’s instructions. T cell purification columns were from R&D.