Fitc conjugated anti mouse cd206 antibody

Flow cytometry was performed to determine the phenotypes of macrophages by examining the expression levels of the M1 marker CD80 and the M2 marker CD206. After seeding on different membranes for 72 h, LPS-treated RAW264.7 cells were digested, rinsed twice with PBS, and then incubated with FITC-conjugated anti-mouse CD80 antibody (1:50; BioLegend) and FITC-conjugated anti-mouse CD206 antibody (1:400; BioLegend) respectively. The labeled cells were then rinsed twice with PBS and examined by a flow cytometer (BD Accuri C6, BD Biosciences, USA). FlowJo 10.0 software was used for the quantitative analysis.

The electrospun nanofiber membranes were first placed in 6-well plates, and RAW264.7 cells were seeded on their surfaces at a density of 2 × 10⁵ cells per well. Following the incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove debris and non-adherent cells. Lipopolysaccharide (LPS, TargetMol) was then added to the culture to activate macrophages. After another 24 h, cells were collected for analysis.
The cell pellets were resuspended in 100 μL of PBS containing 1.25 μg of PE-conjugated anti-mouse CD86 antibody and 0.25 μg of FITC-conjugated anti-mouse CD206 antibody (Biolegend). This step was performed on ice for 30 min to preserve cell viability and prevent nonspecific antibody binding. The expression of CD86 and CD206 was subsequently analyzed using the CytoFLEX flow cytometry system (Beckman Coulter). Additionally, the intracellular ROS level was detected using 2,7dichlorodihydrofluorescein diacetate (DCFH-DA; Yeasen), following the same procedure as described above (n = 3).