Anti tlr4 pe cy7

Monocytes subsets were identified by size, granularity, and by expression levels of CD14 and CD16. Fluorescence minus 1 and isotype gating strategies were used to identify expression of surface markers as previously reported¹⁷ .
Cell surface molecule expression was monitored using the following fluorochrome-labeled antibodies: anti-Tissue Factor fluorescein isothiocyanate (FITC) (American Diagnostica, Stamford, CT), anti-CD14 Pacific Blue, anti-CD16 phycoerythrin (PE), anti-CD36 allophycocyanin (APC), (BD Pharmingen, San Diego, CA), and anti-HLA-DR APC-cy7 (BD Biosciences, San Jose, CA), anti-Toll-like receptor (TLR) 2 APC, anti-TLR4 PEcy7, and anti-TLR-6 biotin (eBioscience, San Diego, CA), streptavidin APC-cy7 (BD Bioscience).
Whole blood samples were incubated for 15 minutes on ice with FACS Lyse buffer (BD Biosciences) and then washed in buffer (phosphate buffered saline with 1% bovine serum albumin and 0.1% sodium azide). Cells were then stained for 30 minutes in the dark on ice and then washed in buffer, fixed in 1% formaldehyde, and analyzed using a Miltenyi MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). MACS Quant software (version 2.21031.1, Miltenyi Biotec), and Prism 5.0 Graphpad software (La Jolla, CA) were used to organize and analyze the data.