Percp efluor labeled anti mouse cd40

PBL and peritoneal macrophages of mice were washed with phosphate-buffered saline (PBS) and stained with PerCP-eFluor-labeled anti-mouse CD40, eFluor 660-labeled anti-mouse CD83, PE-Cyanine7-labeled anti-mouse CD80, and FITC-labeled anti-mouse CD86 (eBioscience, San Diego, CA, USA) at 4°C for 30 min in the dark. For identification of regulatory T cells (Treg cells), isolated PBL was stained with PE-Cyanine7-labeled anti-mouse CD4 and PE-labeled anti-mouse CD25 for 30 min at 4°C in the dark. For intranuclear detection of Foxp3, an anti-mouse Foxp3 staining kit (eBioscience, San Diego, CA, USA) was used, according to the manufacturer's instructions. Briefly, cells were fixed using Fix/Perm buffer and, after washing with 1x permeabilization buffer, were incubated with PE-Cyanine5-labeled anti-mouse Foxp3 Ab for 30 min at 4°C in the dark. Stained cells were analyzed by a FACS Aria III flow cytometer (Becton Dickinson, San Jose, CA, USA) with ≥10,000 gated cells.

Mice were euthanized and peripheral blood leukocytes (PBLs) were collected by cardiac puncture and peritoneal macrophages (pMQs) were isolated from the peritoneal cavity. PBLs were washed with ammonium-chloride-potassium (ACK) lysis buffer to lyse red blood cells. pMQs and PBLs were washed with phosphate buffered saline (PBS). Then 1x10⁶ cells were stained with Percp-eFluor-labeled anti-mouse CD40, eFluor 660-labeled anti-mouse CD83, PE-Cyanine7-labeled anti-mouse CD80, and FITC-labeled anti-mouse CD86 (eBioscience, San Diego, CA, USA) at 4°C in the dark for 30 min. Stained cells were analyzed with FACS Aria III flow cytometer (Becton Dickinson, San Jose, CA, USA) with x10,000 gated cells.