Pe anti mouse cd45 antibody

To investigate the in vivo drug effects, flow cytometry was used to analyze the apoptosis changes and detect myeloid-derived suppressor cells (MDSCs) in the abdomen. On the 48th day after the 1st drug administration, the mice were then sacrificed, ascites or peritoneal washing fluid were collected, and the pellet was separated via centrifuging. An Annexin V/PI Apoptosis Kit was used to analyze apoptosis changes and compare differences in each treatment group. PE anti-mouse CD45 antibody (BioLegend, San Diego, CA, USA, 103,106/200 µg), APC anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (BioLegend, 108,412/100 µg) and FITC anti-mouse/human CD11b antibody (BioLegend, 101,206/500 µg) were used as antibodies to detect MDSCs. Then, samples were subjected to flow cytometry analysis under the guidance of the manufacturer’s protocol.
Ascites, peritoneal washing fluid and tumor tissues from different treatment groups were stained using Caspase 3 Polyclonal antibody and Anti-VEGFA antibody to detect caspase 3 and VEGFA via IHC (methods described in Section 2.2).

Xenografts tumors were mechanically disrupted and digested into single-cell suspension by collagenase IV (Cat: A004186, Sangon Biotech, Shanghai, China) and DNase I (Cat: EN0521, Thermo Fisher Scientific, USA). A cell strainer with a pore size of 70 μm (Biosharp, Hefei, China) was utilized to filter out impurities. T cells in the supernatant were collected for flow antibody staining as follows: PE anti-mouse CD45 antibody (Cat: 147712, Biolegend, USA), PE/Cyanine7 anti-mouse CD3 antibody (Cat: 100220, Biolegend, USA), FITC anti-mouse CD8a antibody (Cat: 100706, Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD4 antibody (Cat. No. 116012, Biolegend, USA), Zombie Aqua Fixable Viability Kit (Cat: 423101, Biolegend, USA).

C57BL6J wild type males (Charles River) from 8 to 12 weeks old were used in this study. Animals were housed in a normal light cycle with ad libitum access to food and water. Hindlimb ischemia experiments were performed as described [15 (link)]. Briefly, mice were anesthetized with isoflurane. The hindlimb was shaved, and the skin was incised. The proximal end of the femoral artery and the distal portion of the saphenous artery were ligated. The artery and all side-branches were dissected to be freed and the femoral artery and attached side-branches were excised. Procedures were approved by the ethics committee (number ZH050-2021). To label circulating b(lood)-CD45⁺ cells, mice were anaesthetised with a combination of ketamine and xylazine and i.v. Injected with 3 μg of PE anti-mouse CD45 antibody (Biolegend [30-F11], 103106) diluted in PBS (as described in Mysore et al. [24 (link)] with minor modifications). Five minutes later, mice were sacrificed and muscle was processed for further analysis (see below). Immediately after sacrifice, blood samples were collected via intracardiac puncture. Blood was directly diluted in PBS containing heparin and was used as positive control for CD45 staining.