Anti pd l1 apc

The 4T1-luc2 tumor cells were treated with crassolide (50 to 3.125 µM) for 24 h. Aliquots of 1 × 10⁶ 4T1-luc2 tumor cells were harvested, washed with PBS, and stained with PE anti-HSP90 antibody (LSBio, LS-63265), Alexa Fluor 488 anti-Hsp70 antibody (BioLegend, #648004), PE anti-HMGB1 antibody (BioLegend, #651404), PE anti-calreticulin (Cell signaling, #62304), APC anti-PD-L1 (BioLegend, #124312), Alexa Fluor 488 anti-CD24 (BioLegend, #101816) in 0.5% bovine serum albumin (BSA, Bionovas) in PBS, and then analyzed using flow cytometry (BD FACSVerse, San Jose, CA, USA).

Five days post-transfection, cells were lifted from culture plates in FACS buffer (ThermoFisher Scientific). Cells were stained with APC-anti-PD-L1 (1:250, BioLegend, #329708). Data from 10,000–15,000 cells were collected on a Guava easyCyte 12HT (EMD Millipore) flow cytometer. FMO was used to determine the positive gate. PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Mononuclear cells (1 × 10⁶ cells) isolated previously from PB, PF, and tumor tissue were incubated with fluorochrome-labeled monoclonal antibodies (mAbs) against cell-surface markers: anti-BDCA-1 FITC (MACS Miltenyi), anti-CD19 PerCP-Cy5.5 (BD Pharmingen), anti-BDCA-2 FITC (MACS Miltenyi), anti-CD123 PE-Cy7 (Biolegend), anti-CD45 FITC (BD Pharmingen), anti-CD14 PE-Cy7 (BD Pharmingen), anti-PD-L1 APC (Biolegend), and anti-PD-L2 PE (Biolegend) for 20 min at room temperature. Then, the cells were washed twice with PBS, and the percentage of myeloid BDCA-1⁺CD19⁻ DCs and plasmacytoid BDCA-2⁺CD123⁺ DCs, and CD45⁺CD14⁺ MO/MA with PD-L1 or PD-L2 expression was analyzed using flow cytometry (FACSCanto I Becton Dickinson, USA). The frequency of mDCs, pDCs, and MO/MA are presented as the percentage of mononuclear cells. For each tube, 100,000 events were acquired and analyzed using FacsDiva software. The expression levels of PD-L1/PD-L2 are presented as the percentage of total respective cell subsets (i.e., myeloid BDCA-1⁺CD19⁻, plasmacytoid BDCA-2⁺CD123⁺ DCs, and CD45⁺CD14⁺ MO/MA). The method of identification pDCs with PD-L1/PD-L2 expression is presented in Figure 13.