Pe conjugated anti mouse cd206

Conditioned medium (CM) was collected from monolayer ADSCs and ADSC spheroid cultures after 1 day and subjected to filtration (0.22 μm filter). RAW 264.7 mouse macrophages were cultured in high-glucose DMEM containing 10% FBS and 1% penicillin-streptomycin. For the inflammatory assay, RAW 264.7 cells were seeded at 25,000 cells/cm² onto 6-well culture plates. After 1 day, monolayer-CM and spheroid-CM were added to the macrophage culture medium at 1 : 3 dilution, respectively. After 48 h culture, mouse macrophages were washed with PBS and harvested for RNA to quantify Arg-1 and IL-10 expression levels by qPCR. For flow cytometry analysis, M2 macrophages were identified as CD206-positive cells. The cells were stained with PE-conjugated anti-mouse CD206 (BioLegend) according to the manufacturer's guide. The percentage of cells staining positive for CD206 was determined by comparing the test samples to macrophages labeled with isotype control antibody. After staining, flow cytometry was performed with a BD LSR II flow cytometer.

The CD86⁺ and CD206⁺ macrophages were detected by flow cytometry. After RAW264.7 cells were cultured for 3 days, macrophages were collected, incubated with phycoerythrin (PE)-conjugated anti-mouse CD206 and PerCP-conjugated anti-mouse CD86 antibodies (Biolegend) for 30 min in the dark, and then washed with PBS to stain cells. They were further incubated with allophycocyanin (APC)-conjugated F4/80 antibodis for 30 min at 4 °C, washed with PBS and detected by flow cytometry (Becton,Dickinson and Company, BD, Franklin Lakes, NJ, USA). Data were analyzed by Flow jo 10.

The expression of cell surface markers and intracellular molecules of macrophages was determined using flow cytometry. Cells were incubated with Fc Block (BioLegend; Cat: 101302; Clone name: 93; Dilution: 1:100) and washed using PBS with 2% fetal calf serum, and then cells were stained with PE-conjugated anti-mouse CD11b (BioLegend; Cat: 101207; Clone name: M1/70; Dilution: 1:100), PE-conjugated anti-TREM2 (R&D Systems, Cat: FAB17291P; Dilution: 1:100), PE-conjugated anti-mouse CD11c (BioLegend; Cat: 117308; Clone name: N418; Dilution: 1:100), PE-conjugated anti-mouse CD206 (BioLegend; Cat: 141706; Clone name: C068C2; Dilution: 1:100), APC-conjugated anti-mouse F4/80 (BioLegend; Cat: 123116; Clone name: BM8; Dilution: 1:100), or isotype antibodies. According to the manufacturer’s instructions, the intracellular staining of KLF9 ((Biorbyt; Cat: orb9122; Dilution: 1:100)) was performed with the transcription factor staining buffer set (eBioscience). Cells were next subjected to flow cytometry analysis with a BD FACSAria IIu flow cytometer (BD Bioscience). Data were analyzed with FlowJo Software version 7.6.4.