Cytokine analyses

Tumor cytokines were quantified by either luminex or by ELISA. Luminex analysis was performed using the Cytokine and Chemokine 36-Plex Mouse ProcartaPlex Panel 1a (Cat# EPX360-26092-901) array. Tumors were collected in 1 mL PBS and disrupted with manual and GentleMacs (Miltenyi Biotec) dissociation. TDLNs were collected similarly in 1 mL PBS and dissociated with syringe plungers and then spun to remove cellular matter. Lysates for luminex analysis were taken directly from the 1 mL solution and plated according to the manufacturer’s suggestions. To reduce luminex sample volume, the DropArray LT210 Washing Station MX (Cat# LT-210MX-01-01; Curiox) was used with microbead plates (Curiox, Cat# 96-CC-BD-05). These plates were then run on using the Bio-Plex 200 (Bio-Rad, Cat#171-000201).

Material for ELISAs was prepared by resecting tumors and dissociating in Cell Lytic M (Sigma; Cat# C2978) with Protease Inhibitor Cocktail (Sigma; Cat# S8820). Tumors were incubated at room temperature for 10 minutes, vortexed, and centrifuged at 10,000 × g to pellet DNA and cellular matter. Lysates were removed and frozen at −80°C until ELISA analysis. The following ELISA assay kits were utilized: mouse IL6 (Cat# M6000B; R&D), mouse TNFα (Cat# MTA00B; R&D), VeriKine mouse IFNβ (Cat# 42400-2; PBL Assay Science), Mouse Inflammatory Cytokines Multi-Analyte ELISArray Kit (Cat# MEM004a; Qiagen).