Collagenase d

Lymph node and brain tissues were digested with 2.5 mg/mL Collagenase D (Roche), 50 μg/mL DNAse I (Roche), and 0.4× protease inhibitor (Roche) in digestion buffer at 37 °C. Digestion of LNs was adapted from Fletcher et al. [29 (link)], such that tissues were digested for 20 min without protease inhibitor followed by three 10-min incubations with protease inhibitor. Brain tissues were digested for 15–20 min at 37 °C. Enrichment of ECs was performed by centrifugation at 5000 g for 30 min at 4 °C in a dextran solution (17% dextran (Sigma, catalog number 31392)/20 mM HEPES). Skin, colon, and small intestine tissues were digested with 2.5 mg/mL Collagenase D (Roche), 50 μg/mL DNAse I (Roche), and 1× protease inhibitor (Roche) in digestion buffer for 30 min at 37 °C on a rotisserie wheel. Colon and small intestine were washed with 5% FBS and 25 mM HEPES, followed by 2 mM EDTA and 25 mM HEPES, and finally 10% FBS, 5 mM EDTA, and 15 mM HEPES prior to enzymatic digestion to remove epithelial cells. Pancreatic and adipose tissues were digested with 1.25 mg/mL Collagenase D (Roche), 50 μg/mL DNAse I (Roche), and 1× protease inhibitor (Roche) in digestion buffer for 30 min at 37 °C on a rotisserie wheel. Cells were resuspended in FACS buffer (PBS (Lonza), 5% FBS (Invitrogen-Gibco), 5 mM EDTA (Boston BioProducts)) for analysis.

Spleen and MLN cells suspension were made by cutting the spleen or MLN with scissors, followed by digestion using 1 mg/mL Collagenase D (Roche) and 50 µg/mL DNase I (Roche) at 37 °C for 45 min. The cell suspensions were filtered through a 70-μm cell strainer (BD Biosciences), red blood cells lysed by the ammonium-chloride-potassium (ACK) buffer (Thermo Fisher) at RT for 5 min and the cells were resuspended in PBS + 10% FBS for further analysis. Colons were dissected, and the stool removed by washing two times with HBSS 1X without Ca²⁺ and Mg²⁺ and finally opened longitudinally. After that, the colon was cut into 0.5 cm pieces, washed with HBSS 1X without Ca²⁺ and Mg²⁺ and incubated in IEL medium (IMDM containing 2% FBS and 1 M HEPES) at 37 °C for 30 min, while constantly stirring. Colon pieces were washed using a 70-μm cell strainer, followed digestion using 1 mg/mL Collagenase D (Roche) and 0.25 mg/mL DNase I (Roche) in IEL medium at 37 °C for 45 min while constantly stirring. Digested tissue was passed through a 70-μm cell strainer obtaining a single cell suspension that was subjected to centrifugation in a Percoll gradient (67%/44%). Mononuclear cells were removed from the interphase and resuspended in culture medium for further analysis.

Tumors were minced and digested in HBSS with Collagenase D (1 mg/mL, Roche) or Collagenase A (2mg/ml, Roche) and DNAse I (50 μg/mL, Roche) for 30 minutes at 37°C in a water bath. Digested tissue was then passed through a 70 μm cell strainer, followed by red blood cell lysis with RBC lysis buffer (Biolegend). Single cells were kept on ice.
For whole intratumoral immune cell profiling and DC stainings, CD45-biotin magnetic positive selection (MACS, Miltenyi) was performed to enrich for total tumor immune infiltrate.
For intratumoral T cell stainings, hematopoietic cells were enriched by density gradient centrifugation with 40/70 Percoll (GE Healthcare) for 20 min at 700xg.
Tumor draining lymph nodes (tdLNs) and spleens were minced and digested in RPMI 1640 with Collagenase D (1 mg/mL, Roche) and DNAse I (50 μg/mL, Roche) for 45 minutes at 37°C in a water bath. Digested tissue was then passed through a 70 μm cell strainer and single cells were kept on ice. For spleens, red blood cells were lysed using RBC lysis buffer (Biolegend).