Pe labeled cd8a

Thymopentin (TP5) was purchased from Meilunbio. Doxorubicin hydrochloride (DOX) was obtained from Aladdin. Fetal bovine serum albumin (FBS) was purchased from Biowest. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco. All assay kits, if not specified, were acquired from Beyotime. The antibodies used were as follows: anti-calreticulin, HMGB1, and CD8 were purchased from Santa Cruz Biotechnology; Ki67 was purchased from Bioss; anti-mouse antibodies (FITC-labeled CD3, FITC-labeled CD11c, FITC-labeled CD25, PE-labeled CD8a, PE-labeled CD86, APC-labeled CD4, APC-labeled CD80, Alexa Flour 647-labeled Foxp3) were all purchased from BioLegend. D-Luciferin, Potassium Salt was purchased from Yeasen.

Tumors and spleens were collected from the B16-F10-bearing mice 21 days post-tumor cell transplantation. The tumor tissue was cut into pieces and digested with 1 mg/mL collagenase IV and 150 U/mL DNase I in RPMI-1640 for 1 h at 37 °C with shaking. The spleen tissue was passed through a mesh to collect single cells. After lysis of the red blood cells, 1.0 µg anti-mouse CD16/CD32 (BD Pharmingen) in 2.0% FBS was added for 10 min to block endogenous non-specific binding to Fc receptors. Mononucleated cells (5×10⁶ cells) were combined with fluorescein isothiocyanate (FITC)-labeled CD8a, phycoerythrin (PE)-labeled CD45, allophycocyanin labeled-CD3, pacific blue-labeled CD4 and PE-CY7 CD11b (Biolegend, San Diego, CA, USA) for 30 min at room temperature. In the β2-AR staining setting, mononucleated cells were incubated with rabbit anti-β2-AR antibodies (Santa Cruz, Dallas, TX, USA), allophycocyanin labeled-CD3, PE-labeled CD8a, and pacific blue-labeled CD4 (Biolegend, San Diego, CA, USA) for 30 min at room temperature and then stained with FITC-goat anti-rabbit IgG F(ab')2 fragments (Jackson Immunoresearch, West Grove, PA, USA) for 30 min. Finally, the fluorescence of the cell suspension was assessed with a Gallios (Beckman Coulter, CA, USA), using Kaluza software (Beckman Coulter) for data analysis.