Collagenase type 1 and 4

For tumor samples, we used methods that were recently described^{44 (link)}. In brief, tumors were trimmed, sliced into small pieces, and digested for 45–95 min with shaking. The enzymatic cocktail for tumor digestion consisted of serum-free Hyclone Leibovitz L-15 medium supplemented with 1% penicillin-streptomycin, collagenase type I and IV (170 mg/L = 45–60 U/mL), collagenase type II (56 mg/L = 15–20 U/mL), DNase I (25 mg/L) and elastase (25 mg/L), all from Worthington Biochemical.

Spleens were harvested in LPA Media (without BME), dissociated through 70-mm filters, and subjected to ACK lysis (Quality Biological) to obtain splenocytes for analysis. Tumors were harvested in RPMI with 5% FBS media, cut into small pieces, and incubated for 30 min at 37ºC and 300 rpm in a digestion cocktail composed of RPMI supplemented with 5% (v/v) FBS, 2 mg/mL Collagenase Type I and IV (Worthington Biochemical Corporation), and 40 U/mL DNase I (Calbiochem). Following digestion, tumors were ground through 70-mm filters, spun for 5 min at 500 g, and resuspended in media for cell counting and subsequent flow cytometry analysis. Mice serum was collected into serum tubes with separating gel (BD BioSciences), rested for 30 min, and centrifuged at 2000 rpm for 2 min. Supernatant was stored at − 20 ºC until analysis.
Lungs were collected, rinsed with PBS 1 × , and weighed. They were then transferred to gentleMACS M tubes (Miltenyi Biotec) and the appropriate amount of PBS 1X was added based on weight (80 μL of PBS 1 × for every 25 mg of tissue). The tissue was mechanically disrupted using the gentleMacs dissociator (Miltenyi Biotec), and 80 μL aliquots of tissue homogenate were stored in DNAse/RNAse free tubes at  − 80 ºC until further processing.