Mouse cell depletion purification kit

When triple negative breast cancer PDX tumors grew to 1cm in diameter, tumors were dissected, minced and then incubated with the enzyme mix and from a human Tumor Dissociation Kit (Miltenyi Biotech). Human breast cancer tumor cells were isolated using the Mouse Cell Depletion Purification Kit (Miltenyi Biotech) following the manufacturer’s protocol. The purified breast cancer cells were transduced with control or PD-L1 shRNA lentivirus for 2 hours. The transduced cells were seed in 96 well plate and irradiated with indicated dose of ionizing radiation at the following day. Cell spheroids were counted using a Nikon Eclipse Ts100 (Nikon Instruments Inc.), and the susceptibility of the cells to IR was investigated using the percentage of viability.

PDX #4 and #14 cells were purified using the protocol described previously (Gao et al., 2014 (link)). In brief, a portion of tumor tissue was dissected and cut into 2 mm³ sections in sterile PBS and incubated with 5 ml of the enzyme mix from a human Tumor Dissociation Kit (Miltenyi Biotech) prepared according to the manufacturer’s protocol. Tissue was digested at 37°C with rotation on a gentleMACS Dissociator (Miltenyi Biotech) program for “dissociation of tough tumors” according to the manufacturer’s protocol. The digested cell mixture was passed through a 70 μm MACS SmartStrainer, then a 40 μm MACS SmartStrainer to obtain the single-cell suspension. Dissociated cells were then spun down and washed with precooled washing buffer. Purified breast cancer tumor cells were obtained by removing the mouse cells using the Mouse Cell Depletion Purification Kit (Miltenyi Biotech) according to the manufacturer’s protocol. The obtained PDX cells were cultured in DMEM supplemented with 10% FBS, glutamax and NEA, infected with a lentivirus encoding human MtCK1 shRNA construct (TRCN0000006057), and selected with 1 μg/mL puromycin before tumor formation and cell proliferation assay started. For cell proliferation assay, CyQUANT Cell Proliferation Assay Kit (Thermo Fisher Scientific) was used as described in the above section.