Percpcy5.5 conjugated f4 80

Cells (1 × 10⁶) were stained with antibodies for 30 min in dark before analyzed in a FACS Canto cytometer (BD Biosciences, Franklin Lakes, NJ). For intranuclear staining, cells were fixed and permeabilized using Foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA) and stained with Ki67. The antibodies used included PE-conjugated PD-1L, PerCPCy5.5-conjugated F4/80, APC-conjugated CD11b (Mac-1), CD44 and CD86 (Biolegend). For cell cycle, cells were fixed overnight in 70% ice-cold ethanol at –80 °C, as described86 (link). Cells were stained with propidium iodide/ribonuclease A (RNaseA) solution (BD Biosciences) for 30 min before analyzed using flow cytometer.

The following fluorochrome-conjugated monoclonal antibodies were used in this study: anti-mouse PE/Cy7-conjugated CD45 (Cat# 940335), APC-conjugated Ly6G (Cat# 560599), PE-conjugated CD11b (Cat# 553311), PE-conjugated CD19 (Cat# 553786), and APC/Cy7-conjugated NK1.1(Cat# 560618) (BD Biosciences, San Diego, CA, USA). PerCP/Cy5.5-conjugated F4/80 (Cat# 157317), APC/Cy7-conjugated Ly6C (Cat# 128025), FITC-conjugated CD14 (Cat# 123308), (Biolegend, San Diego, CA, USA), and PE/Cy7-conjugated CD3 (Cat# 25-0031-82) (eBioscience, San Diego, CA, USA). Cells were blocked with FcR blocker (BD Pharmingen) and then incubated with the indicated antibodies under standard protocols. All samples were analyzed using flow cytometry (FACSVerse system, BD Biosciences) with FlowJo (version 7.6.1) software.