Fitc conjugated anti cd8a

To examine the surface expression of PDPN in mouse tumor cell lines, tumor cells were incubated with PMab‐1‐mG_2a or isotype control (1 μg/mL) for 30 min. Cells were washed with PBS and incubated for 30 min with FITC‐conjugated goat F(ab’)₂ fragment anti‐rat IgG (H + L) Ab (Beckman Coulter). AB1‐HA tumor tissue was cut into small pieces with scissors and digested by digestion buffer consisting of 1 mg/mL BSA (Sigma‐Aldrich), 1 mg/mL collagenase IV (Thermo Fisher Scientific Inc.), and 100 μg/mL DNase I (Roche) in DMEM for 40 min of incubation at 37°C, followed by passaging through a 100 μm cell strainer. The cells were incubated with FcR blocking reagent (BD Biosciences). To examine the surface expression of CTLA‐4 and PD‐1 in mouse tumor‐infiltrating immune cells, tumor tissue‐derived cells were stained with FITC‐conjugated anti‐NKp46 (BioLegend), FITC‐conjugated anti‐CD8a (BD Biosciences), FITC‐conjugated anti‐CD4 (BD Biosciences), FITC‐conjugated anti‐F4/80 (BioLegend), PE‐Cy7‐conjugated anti‐CTLA‐4 (BioLegend), and PE‐Cy7‐conjugated anti‐PD‐1 (BioLegend). The stained cells were analyzed by flow cytometry using a BD LSRFortessa (BD Biosciences) for acquisition and the FlowJo software program (Treestar Inc.) for the analysis.

pX330 plasmid was obtained from Addgene (#42230). pMax-GFP plasmid was acquired from Amaxa (Lonza, Basel, Switzerland). Antibodies used for Western blot were from as the following, Cdk5 (Santa Cruz Biotechnology, SC-6247, Delaware Ave, USA), p35 (Santa Cruz Biotechnology, SC293184), β-actin (Cell Signaling, 4967S, Danvers, USA). Cdk5 (Abcam, ab40773, Cambridge, UK) and PD-L1 (Abcam, ab205921) antibodies were obtainedfor immunostaining. The in vivo anti-mouse PD-L1 (B7-H1) antibody was purchased from BioXcell, USA (BP0101). The antibodies for flow cytometry were purchased from BD Pharmingen™, including PE Rat anti-mouse CD274 (558091), PE-Cy5-conjugated anti-CD3e (553065), PE-conjugated anti-CD4 (553730) and FITC-conjugated anti-CD8a (553030). Treg kit was purchased from Novusbio, USA (MAGM208). (1,4-butanediol) BDD, (5-amino-1-pentanol) AP and (1-(3-aminopropyl)-4-methylpiperazine) AMP were obtained from TCI, Shanghai, China. All the solvents used were of analytical grade and produced by Sinopharm, Beijing, China. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cre), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).