Fitc conjugated anti cd11b m1 70

Mouse bone-marrow-derived macrophages were washed, incubated with anti-Fc receptor (αCD16/32, clone 93, eBioscience, San Diego, CA) and analysed by flow cytometry using the following antibodies: phycoerythrin-conjugated anti-SIRPα (P84, eBioscience), allophycocyanin-conjugated anti-F4/80 (Cl:A3-1, AbD Serotec, Kidlington, UK), FITC-conjugated anti-CD11b (M1/70, Biolegend, San Diego, CA), peridinin chlorophyll protein (PerCP) -conjugated anti-MHCII (AF6-120·1, Biolegend) and hCD47-Fc or mCD47-Fc plus FITC-conjugated anti-human IgG Fcγ (Jackson ImmunoResearch, West Grove, PA). Phycoerythrin-conjugated rat IgG1 negative control (AbD Serotec), allophycocyanin-conjugated rat IgG2b negative control (AbD Serotec) and mCD7 (aglycosyl)-Fc (isotype matched) were used as controls. Flow cytometry was conducted with a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and data were analysed using flowjo (Tree Star Inc., Ashland, OR).

The pooled endometriotic lesions from each mouse were processed to become single-cell suspensions. The lesions were incubated in 0.05% trypsin, 0.1% collagenase D, 0.53 mmol/L EDTA, and 150 mg/mL DNase I for 40 min and were then mechanically disrupted with a gentle MACS dissociator (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. The lesion single-cell suspensions were stained with PerCP/Cy5.5-conjugated anti-CD45 (30-F11, BD Biosciences), APC-conjugated anti-CD11c (N418, eBioscience, San Diego, CA, USA), PE-conjugated anti-PDCA-1 (eBio927, eBioscience), PerCP/Cy5.5-conjugated anti-Ly6C (HK1.4, BioLegend), BV421-conjugated anti-F4/80 (T45-2342, BD Biosciences), FITC-conjugated anti-CD11b (M1/70, BioLegend), Alexa Fluor 700-conjugated anti-CD44 (IM7, BioLegend), PE-conjugated anti-CD45 (30-F11, BD Biosciences), and FITC-conjugated anti-ICAM-1 (YN1/1.7.4, BioLegend).

Single-cell spleen suspensions obtained from WT and TG mice were prepared by treatment with red blood cell lysis solution (0.15 M NH₄Cl and 0.1 mM Na₂EDTA) for 30 min at room temperature. Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (M1/70, Biolegend, CA, USA), FITC-anti-CD4 (GK1.5, BD Biosciences, CA, USA), FITC-anti-F4/80 (BM8, Thermo Fisher, MA, USA), phycoerythrin (PE)-conjugated anti-CD11b (M1/70, Biolegend), PE-conjugated anti-Ly6G (RB6-8C5, Thermo Fisher), APC-anti-Ly6C (HK1.4, Thermo Fisher) and Alexa Fluor 700-anti-CD11c (N418, Thermo Fisher) antibodies were used. Isotype control antibodies were used optimally.
All samples were acquired and analysed using a FACSAria™ III flow cytometer (BD Biosciences) and a BD Accuri C6 flow cytometer (BD Biosciences).