Pd l1 antibody

Tumor cells were washed with PBS and stained with phycoerythrin (PE)‐conjuncted PD‐L1 antibody (29E.2A3, Biolegend) and then determined with flow cytometry (Beckman Coulter). Data were analyzed using Flow Jo software (Tree Star).

Murine CD64 antibody for western blot and HPR labeled secondary antibody were from R&D Systems and KPL, respectively. GAPDH antibody was from Abmart. CD8 and CD4 antibodies for immunohistochemical staining and Alex Fluor 647 and Alex Fluor 488 labeled secondary antibody were purchased from Abcam. CD25, Ki67, Foxp3 antibodies for flow cytometry analysis and PD-L1 antibody for western blot were from Biolegend Inc. FITC-CD3 antibody was from Ebioscience. PE-CD4 and APC-CD8 antibodies were purchased from Life. Cyclophosphamide and hygromycin were from Sigma-Aldrich. Wheat Germ Agglutinin (WGA) Alexa Fluor 488 and 594 dyes were from Thermo Fisher.

PAT of control mice or mice subjected to LAD were collected after the sacrifice of the mice and perfusion with PBS to remove peripheral cells. PAT was then finely minced and digested in collagenase A solution (2.5 g/L collagenase A and 50 μg/ml DNase (Roche, Basel, Switzerland)) at 37°C for 45 min. The single cell suspension was rinsed, resuspended in PBS supplemented with 3% FBS and CD16/CD32 Fc‐block (clone 2.4G2; BD Pharmingen, BD Biosciences, Franklin Lakes, NJ, USA) and stained with a mix of fluorochrome‐conjugated antibodies for 45 min on ice. Antibodies used were CD45 (clone 30F11), CD11b (clone M1/70), F4/80 (clone BM8), CD3 (clone 17A2), as well as CD25, Foxp3, CD44, CD62L, CD4, CD8, annexin V for lymphocyte population analysis (all from BioLegend) and CD206 (BioLegend) and MertK (AF591, R&D, Abingdon, UK) for macrophage population analysis. Flow cytometry experiments were also performed using differentiated cADSCs with PD-L1 antibody (BioLegend). Data were acquired on a Fortessa (BD Biosciences, Franklin Lakes, NJ, USA), and analysis was performed with FlowJo software (Ashland, OR, USA).

Fluorescence-based ELISA and cell-based tests were used to examine the binding specificity of PPL-C to PD-L1. To perform the ELISA, fluorescent plates were coated with 100 μL human PD-L1 (hPD-L1), mouse PD-L1 (mPD-L1), and BSA in PBS (pH 7.4) overnight at 4°C, respectively. Blocked the plates with 1% BSA (in PBST) for an hour at room temperature and rinsed twice with PBST. Then added peptides (20 μmol/L FITC-labeled PPL-C/Control peptide) and incubated for 1.5 hours at room temperature. With excitation at 488 nm and emission at 515 nm, the Cytation 3 instrument (BioTek) was used to measure the relative fluorescence intensity. Both attached and detached cells were used in the cell-based flow cytometry analysis. With 0.25% trypsin, cells from the following types were detached: CHO, CHO-PD-L1 (CHO cells transfected with a recombinant human PD-L1 full-length expression plasmid), MDA-MB-231, MC38, CT26, and LLC cells. After that, cells were treated for 40 minutes at 4°C on an inversion shaker with PD-L1 antibody from Biolegend and 4 μmol/L FITC-labeled peptides (PPL-C/Control peptide). Each tube was washed twice with PBS following incubation and flow cytometry was carried out using a FACSort device. FlowJo software was used to evaluate the data that was acquired.