Anti foxp3 percp

The population of each phenotype of immune cells in different groups was evaluated by using flow cytometric analysis as described previously [30 (link)]. In brief, splenocytes were stained with fluorescent antibodies, including anti-CD4-FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-APC, anti-MHCII-FITC, anti-CD86-PE, anti-CD40-PE, anti-CD4-PE (eBiosciences, San Diego, USA), anti-IL-4-APC, anti-CD68-FITC, and anti-CD206-PE (BioLegend, San Diego, USA), according to the manufacturer’s instruction. The FlowJo software was used to analyze the percentages of various phenotypes of immune cells.

The phenotype of various immune cells was evaluated by flow cytometry analysis [FACS, Epic XL, Software Expo32 (Beckman coulter)]. Briefly, splenocytes were stained with fluoresent antibodies, including anti-CD3ε-FITC, anti-CD8a-PerCP, anti-CD4-PE/FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-PE, anti-MHCII-FITC (eBiosciences, San Diego, CA, USA), according to the manufacturer’s instructions. The percentage of each phenotype of immune cells was analyzed with the corresponding Flowjo software.

For analysis of CD4+ and CD8+ T cells, CD28 expression in CD8+ T cells infiltrated and various cytokines in the distal tumor tissues of mice, the inoculation was performed by subcutaneous injection of H22 (2 × 10⁵) cell suspension on bilateral sides of each female ICR mouse (N = 7). After 1 week, the primary tumors of mice were treated by PTT via the intravenous injection of PLOV or surgery. Tumor drainage lymph nodes were harvested from mice in 5th day or 10th day after treatment and ground with the rubber head of a syringe. Cells were filtered through nylon mesh filters and washed with PBS. After adding lymph extract, the cells were centrifuged by gradient centrifugation to obtain a complete lymphocyte suspension. The complete lymphocyte suspension (200 μL) was further stained with anti-CD8-FITC (eBioscience), anti-CD4-FITC (eBioscience), anti-Foxp3-PerCP, anti-CD28-PE (eBioscience) and anti-CD38-FITC (eBioscience) at 0.25 μg/test for 20 min. The cell suspensions mentioned above were collected for data analysis by flow cytometry (BD Biosciences, America).