Collagenase type 2 from clostridium hystoliticum

Before flow cytometry analysis, splenocytes were lysed with ACK lysis buffer (Lonza) and washed with PBS. Isolated tumors were incubated in media with 1 mg/mL Collagenase type II from Clostridium hystoliticum (Sigma) for 1 h and later washed with PBS and counted. Prepared cells were stained with murine antibodies for 1 h at 4⁰C protected from light. The following antibodies were used in the study: Live/Dead-eFluor 506 (eBioscience), Fc block (mCD16/CD32, BioLegend), CD3-APC-Cy7 (BioLegend), CD8a-FITC (BioLegend), CD4-FITC (BioLegend), mFoxP3-APC (eBioscience). All data were collected on a FACSverse flow cytometer and analyzed using CaseViewer software (3DHISTECH Ltd., Budapest, HU).

Mice in the experiment were previously immunized with DNA vaccine coding for OVA protein together with an adjuvant plasmid coding for IL-12 as described in Animal immunization. Tumors were excised 14 days after tumor induction and blood was collected aseptically for further determination of IgG titers. The wounds induced by tumor excision were stitched and target splenocytes were injected intravenously, as described in the In vivo killing assay section. At day 16 after tumor induction, mice were sacrificed and spleens were collected for further analysis. Before further analysis, the single cell suspension was prepared from spleen and tumor tissue. Red blood cells and spleen samples were lysed (ACK lysis buffer, Lonza, Walkersville, USA) and splenocytes were washed with PBS. Isolated tumors were incubated in media with 1 mg/mL Collagenase type II from Clostridium hystoliticum (Sigma) for 1 h and later washed with PBS and counted. In spleen tissue the percentage of antigen-specific killing was determined, in tumors the tetramer assay was performed and in serum samples the immunoglobulin titers were measured.

MSCs were extracted from human adipose tissues of healthy female patients undergoing cosmetic surgery procedures following guidelines from the Clinic of Plastic Surgery, University of Padova. Adipose tissues were digested with 0.075% collagenase type II from Clostridium hystoliticum (Sigma–Aldrich, St Louis, MO, USA) in phosphate-buffered saline (PBS). Floating adipocytes were discarded, and cells from the stromal-vascular fraction were pelleted, rinsed with medium, and centrifuged. MSCs were obtained after a red blood cell lysis step in NH₄Cl for 10 min at room temperature.