Apc conjugated anti cd11c n418

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).

Blocking antibodies anti-Dectin-1 (2A11), anti-Dectin-2 (D2.11E4) and anti-CR3 (5C6) were purchased from Serotec and anti-TLR-2 (6C2), IgG2a (eBR2a) and IgG2b (eB149/10H5) were from eBioscience. PE/Cy7-conjugated anti-CD45 (30-F11), APC-conjugated anti-F4/80 (BM8), Alexa 488-conjugated anti-CD103 (2E7) and Alexa 647-conjugated anti-Ly6G (1A8) were obtained from BioLegend. APC-conjugated anti-CD11c (N418) and PE-conjugated anti-CD11b (M1/70) were from eBioscience and PE-conjugated anti-Siglec-F (E50-2440) from Pharmingen.
Z-YVAD-FMK (Caspase-1 inhibitor) was obtained from BioVision. U1026 (ERK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), N-acetyl-L-cysteine (ROS scavenger), Apocynin (NADPH-oxidase inhibitor), Ca-074 methyl ester (cathepsin B inhibitor), bafilomycin A1 (phagosomal acidification inhibitor) and glibenclamide (K⁺ channel inhibitor) were obtained from Sigma-Aldrich.

Recombinant murine TNF-α (315-01, PeproTech, Rocky Hill, NJ) was used. IKK inhibitor III (also known as BMS-345541; 401480, Calbiochem) and U73122 (662035, Calbiochem) were purchased. Anti-PLCε antibody against the C-terminal peptide of mouse PLCε was in-house generated [17] (link) and capable of reacting with the lipase-dead protein present in PLCε mice [16] (link). Other antibodies used are as follows: Anti-CC chemokine ligand (Ccl)2 (sc-1785, Santa Cruz), anti-chemokine (C-X-C motif) ligand (Cxcl)2 (AF-452-NA, R and D Systems, Minneapolis, MN), PerCP-Cy5.5-conjugated anti-CD45R (RA3-6B2, eBioscience), APC/Cy7-conjugated anti-CD3ε (145-2C11, BioLegend, San Diego, CA), PE-conjugated anti-CD4 (RM4-5, BD Biosciences, San Jose, CA), APC-conjugated anti-CD11c (N418, eBioscience), FITC-conjugated anti-MHC class II (M5/114.15.2, eBioscience), and anti-pan cytokeratin (C-11, abcam). CF dye-labeled secondary antibodies were purchased from Biotium (Hayward, CA).