Collagenase d from clostridium histolyticum

Tumor-bearing mouse lungs were collected from KP mice after which tumor nodules were excised and cut into about 1 mm pieces before placement under Hank’s Balanced Salt Solution (HBSS) containing 100 U/mL Collagenase D from Clostridium histolyticum (Sigma Aldrich) and 50 μg/mL DNase I grade II from bovine pancreas (Sigma Aldrich) for 40 minutes at 37°C. After digestion, cells were passed through a 70 µm strainer to remove clumps, and treated with ACK Lysing Buffer (Life Technologies). Cells were resuspended in FACS buffer (PBS + 2% fetal bovine serum) for flow cytometry. For multicolor flow cytometry, cells were first stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation (Life Technologies) for 30 minutes at 4 °C and washed twice with FACS buffer. Cells were treated with purified anti-mouse CD16/32 (BioLegend) for 15 minutes, and then antibody mixture was added. Thirty minutes later, the cells were washed twice with FACS buffer and fixed in 1% formalin or further processed for intracellular staining. For intracellular staining, cells were fixed/permeabilized with Foxp3/Transcription Factor Staining Buffer Set Kit (eBioscience) before antibodies were added. After two washes, samples were resuspended in FACS buffer before acquisition using BD LSR Fortessa or BD FACS Canto (BD Biosciences)].