Apc anti mouse cd206

Cells were scraped from the culture dish and then washed three times with cold PBS. Staining was performed according to the manufacturer’s recommended protocol using PE anti-mouse CD86 (12-0862-82, Thermo Fisher, USA) and APC anti-mouse CD206 (17–2061-82, Thermo Fisher, USA) antibodies. Data acquisition and further analysis was conducted using the CytExpert software (version 10.0.7; Tree Star, Ashland, OR, USA).

To determine macrophage recruitment and phenotype in BAL, cells were labelled with the following surface markers: PE anti-mouse F4/80 (BioLegend, San Diego, CA), BV650 anti-mouse CD86 (BD, Franklin Lakes, NJ), PE-cy7 anti-mouse iNOS (Thermo Fisher Scientific), APC anti-mouse CD206 (Thermo Fisher Scientific) for 20 min at 4°C. Cells were then washed twice with cold flow cytometry buffer (PBS, 0.5% BSA) and incubated for 30 min at 4°C. Cells were subsequently washed and resuspended with cold flow cytometry buffer for analysis. Isotype-matched control antibodies were used to distinguish the cut-off between negative and positive fluorescent populations. Data were collected by a BD LSRFortessa™ flow cytometer and analyzed using FlowJo V10 software.

BV2 cells and primary microglia were collected after the indicated treatments and stained with fluorescence-conjugated monoclonal antibodies according to the manufacturer’s instructions. Cultured and treated cells were resuspended in PBS solution. Then, a 95-μL cell suspension was obtained, and 5 μL of FITC-anti-mouse CD40 (11–0402-81, Thermo Fisher, USA), APC-anti-mouse CD206 (17–2061-82, Thermo Fisher, USA), or CoraLite® 488 anti-mouse CD11b (CL488-65,055, Proteintech, China) was added. The suspension was incubated for 2 h at 4 °C in the dark, fixed with 2% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 15 min. Finally, the cells were blocked with 1% bovine serum albumin (BSA, BS114-100g, Biosharp, China) for 1 h and washed three times with PBS buffer. Subsequently, 5 μL of PE-anti-mouse CD16/32 (12–0161-82, Thermo Fisher, USA) or PE-anti-mouse Arg-1 (12–3697-82, Thermo Fisher, USA) and CoraLite® 647 anti-mouse MHC Class II (CL647-65,122, Proteintech, China) were added, and the suspensions were incubated at 4 °C for 2 h in the dark. Then, the cells were washed three times with prechilled 1% BSA solution. Finally, flow cytometry was performed using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analysed using FlowJo™ v10 software (for Windows) Version 10 (Ashland, BD Life Sciences).