Apc conjugated anti mouse cd86 antibody

Cell differentiation trends were quantified using flow cytometry. For flow cytometry, the cells (1.0 × 10⁶ per sample) were washed with PBS and incubated with antibody in a blocking solution (1% v/v BSA in PBS) for 30 min. RAW264.7 cells were stained with APC-conjugated anti-mouse CD86 antibody (cat.105011, Biolegend, San Diego, CA, USA) or PE-conjugated anti-mouse CD206 antibody (cat.141705, Biolegend, San Diego, CA, USA) to analyze the Macrophage M1 or M2 frequency, respectively. For Th17 cell analysis, T cells were stained with FITC-conjugated anti-rat CD4 antibody (cat.201505, Biogend, San Diego, CA, USA) and eFluor^TM506 anti-rat IL-17A antibody (cat.69-7177-82, ThermoFisher, Waltham, MA, USA) and stained with PE-conjugated anti-rat CD25 antibody (cat.202105, Biolegend, San Diego, CA, USA) and Alexa Fluor^® 488 anti-rat Foxp3 antibody (cat.320012, Biolegend, San Diego, CA, USA) to analyze Treg cell. Data were analyzed with FlowJo_V×10.8 software (FlowJo LLC, Ashland, OR, USA).

RAW264.7 cells were seeded onto 6-well plates at a concentration of 2 × 10⁵cells/well. After incubation for 24 h at 37 ℃, the medium was removed and washed three times with PBS, followed by the addition of different CM to each well. To mimic the in vivo inflammatory conditions of diabetic wounds and explore the effect of NETs and DNase I on macrophage repolarization, LPS (Sigma-Aldrich), NETs extracts, and DNase I (Sigma-Aldrich) were added into each well, respectively. After a 24-h culture, RAW264.7 cells were collected via centrifugation at 1000 rpm for 5 min, and the pellets were resuspended with 100 μL PBS containing 0.25 μg APC-conjugated anti-mouse CD86 antibody (Biolegend) and 0.5 μg PE-conjugated anti-mouse CD206 antibody (Biolegend), followed by incubation on ice for 30 min. CD86 and CD206 levels on RAW264.7 cells were analysed by flow cytometry using the CytoFLEX system (Beckman Coulter).

RAW264.7 cells were first pre-stimulated with 100 ng/mL of IL-4 for 24 h to polarize them into M2-type macrophages, and then co-incubated with LPS, MENP, or MP- MENP for another 24 h. The cell supernatant was carefully collected for the cytokine secretion assay using ELISA assay kits. Meanwhile, the cells were collected for APC-conjugated anti-mouse CD86 antibody (Biolegend, USA) and 0.5 µg PE-conjugated anti-mouse CD206 antibody (Biolegend, USA) incubation, and then detected using flow cytometer.