Tumor dissociation buffer

PV-10 was provided by Provectus Biopharmaceuticals. Tumor cells were incubated with various doses of PV-10 for the indicated time in vitro and cell supernatant were collected. For generation of tumor explant supernatants (TES), when tumors reached 100-150 mm², 50 ul of PBS or PV-10 were IL injected. After 24 hours, tumors were collected. Single-cell suspensions were obtained by passing cells through cell strainers and cultured at 2 million cells per ml for 24 hours. TES were collected after centrifugation.
For tumor digestion, tumors were isolated from tumor-bearing mice treated with PV-10 or PBS and were digested with tumor dissociation buffer (Miltenyi Biotec) and GentleMACS (Miltenyi Biotec). After lysis of RBCs, single-cell suspensions were analyzed by FACS.

Mice were euthanized and the tumors were harvested and placed in 1 mL of cold FBS free RPMI. The tumors were cut into smaller pieces with a scalpel and 1 mL of Tumor Dissociation Buffer (Miltenyi Biotec, #130-096-730) was added and incubated at 37°C for 40 minutes with continuous shaking at 90 rpm. After the incubation, the dissociation was quenched by the addition of 3 mL of RPMI with 10% FBS and the samples were filtered through a 70 μm filter. After centrifugation at 1,500 rpm for 5 minutes, the supernatant was discarded and the pellet was resuspended in PBS.