Pe conjugated anti pd l1 antibody

For melanoma CTC experiments, 500,000 Mel167 CTC cells were treated with 30 pmol of KEAP1-targeting siRNA smart pools or non-targeting controls (Horizon Discovery) overnight. Media was refreshed the next day, and 48 h after siRNA treatment, cells were collected for qRT-PCR validation of KEAP1 knockdown. Cells were also treated overnight with vehicle control or 100 ng/mL interferon gamma (Cell Signaling Technology #80385). Cells were collected, rinsed twice with PBS containing 2% fetal bovine serum, and incubated on ice for twenty minutes for cell-surface staining with a PE-conjugated anti-PD-L1 antibody (Biolegend #329705, 1:20) or a PE-congugated anti-HLA-A/B/C antibody (Biolegend #311405, 1:20). After two more washes, cells were analyzed by flow cytometry. KEAP1 knockout and control B16-ova cells, described above, were treated overnight with vehicle control or 100 ng/mL interferon gamma (Cell Signaling Technology #39127). Cells were stained with a PE-conjugated anti-PD-L1 antibody (Biolegend #155403, 1:20), as described above, and analyzed using flow cytometry. The expression of the MHC-I HLA gene, H2-d1 was analyzed by qRT-PCR. Antibody dilutions are included in Supplementary Table 2.

The PD-L1 expression on tumor cells was assayed with flow cytometry. After washing with phosphate-buffered saline (BI, Israel), the cells were harvested by enzyme-free cell dissociation buffer (Thermo Fisher, USA). The harvested cells (1.5 × 10⁶) were stained with phycoerythrin (PE)-conjugated anti-PD-L1 antibody (Biolegend, USA), and the PD-L1 expressing cells were quantified by Navias Flow Cytometer (Beckman Coulter, USA).