Apc h7 conjugated anti cd14

Evaluation of the MDSC percentage was accomplished with 0.5*10⁶ cells stained with FITC-conjugated anti-CD15, PE-conjugated anti-CD33, PerCP-conjugated anti-HLA-DR, a cocktail of APC-conjugated antibodies anti-CD3, -CD56, -CD19 (Lin), APC-H7-conjugated anti-CD14 and PE-Cy7-conjugated anti-CD11b (BD Biosciences). Acquisition of 100,000 events was performed in the leukocyte-gated population on FACS CANTO II and analyzed with FACS DIVA software (BD Biosciences, USA).

PBMCs were thawed briefly in a 37°C water bath before washing with DMEM. Cells were then resuspended in phosphate buffered saline (PBS, pH 7.2) and stained for flow cytometry. For intracellular staining, cells were first stained with surface stains, then fixed and permeabilized with foxp3/transcription factor staining buffer (Invitrogen, Waltham, MA) before staining with intracellular stains. Flow cytometry for PBMCs was run on a BD LSR Fortessa™ flow cytometer (BD Biosciences, San Jose California, USA) and data were collected using BD FACSDiva™ software (BD Biosciences, San Jose California, USA). Data were analyzed using FlowJo Version 10 Software (FlowJo, Ashland, Oregon, USA). The following antibodies were used for staining: PE CF594-conjugated anti-CD15; APCH7-conjugated anti-CD14; PE-conjugated anti-CD33; APC-conjugated anti- HLA-DR, FITC-conjugated anti-Lineage; PE/Cy7-conjugated anti-CD123; PE-conjugated anti-TLR7 (all from BD Biosciences, San Jose, CA); A700-conjugated anti-CD11b (Life technologies, Carlsbad, CA); FITC-conjugated anti-TLR7 and FITC-conjugated anti-Rabbit IgG isotype control (both from Invitrogen, Waltham, MA); biotinylated-anti TLR9 (Abcam, Cambridge, UK); unconjugated anti-human FC block, PerCP/Cy5.5-conjugated Streptavidin; biotinylated anti-Mouse IgG2a and PE-conjugated anti-Mouse IgG1κ (all from eBioscience inc, Santa Clara, CA).