Percp cyanine 5.5 labeled anti mfoxp3 antibodies

Blood samples (100 μL) were collected from each group on day 30 after inoculation of tumor cells, and the red blood cells were then lysed by ACK lysing buffer (Solarbio). To detect DCs, cells were stained with FITC‐labeled anti‐mCD11c and PE‐labeled anti‐mCD80 antibody for 30 minutes. To qualify the CD4⁺/CD8⁺ T populations, cells were stained with FITC‐labeled anti‐mCD4 or FITC‐labeled anti‐mCD8 antibody for 30 minutes. To detect the Treg populations in the blood of each experimental group, the cells were incubated with FITC‐labeled anti‐mCD4 for 30 minutes, then the cells were stained with PerCP‐Cyanine 5.5‐labeled anti‐mFoxP3 antibodies for 30 minutes after fixed and permeabilized using the Foxp3 staining kit (eBioscience). T‐cell subsets were measured by flow cytometry (Becton Dickinson).

Tumor tissues were obtained from 20 PCa patients and normal tissues were selected from 10 patients who received pathological biopsy of benign disease in Nanfang Hospital (Guangzhou, China). None of these patients had received immune‐regulating drugs or radiation therapy before surgery. The study was approved by the ethics committee and informed consent was obtained from all patients. Norcantharidin, CCK‐8, and DAPI reagents were purchased from Sigma (St. Louis, MO, USA). Streptavidin‐tagged mouse GM‐CSF (SA‐mGM‐CSF) fusion protein was prepared by our laboratory. Western blotting antibodies against p‐ERK1/2, p‐Akt, ERK1/2, Akt, Bcl‐2, FOXO1, FasL, and GAPDH were purchased from Abcam (Cambridge, UK). Phycoerythrin‐labeled anti‐mGM‐CSF, FITC‐labeled anti‐mCD11c, PE‐labeled anti‐mCD80, FITC‐labeled anti‐mCD4, FITC‐labeled anti‐mCD8 and PerCP‐Cyanine 5.5‐labeled anti‐mFoxP3 antibodies were purchased from eBioscience (San Diego, CA, USA). C57BL/6 male mice (6‐8 weeks old) were purchased from the experimental animal center of Southern Medical University (Guangzhou, China). All animal studies were undertaken in accordance with the Southern Medical University guidelines for experimental animals.