Cd15 pe cy7

The expression of TLR4, NOX2, and CD11b on the surface of neutrophils and monocytes was evaluated by flow cytometry. Whole blood (200 μL) was treated in 100-μL aliquots incubated at 37°C for 1 h untreated (vehicle) or with 10 ng/mL LPS (SIGMA Life Science, Ireland). Fluorochrome-conjugated monoclonal antibodies (mAb) specific for humans CD14-PerCP, CD15-PECy7, NOX2-FITC, CD66b-Pacific Blue, TLR4-APC (BioLegend®, USA), and CD11b-PE (BD Biosciences, UK) were used. The whole blood was then stained with mAb for 15 min. Red blood cells were lysed with BD lysis buffer. Cells were acquired on a FACSCanto II flow cytometer (BD Bioscience) and analyzed using FlowJo version 10 (Tree Star) (2 (link), 14 (link)). Neutrophils were delineated based on SSC-A and CD66b⁺ and monocytes based on SSC-A, CD66b⁻, and CD14⁺ (15 (link), 16 (link)). Monocytes were subdivided into classical, intermediate, and non-classical subtypes. Monocyte subsets were identified as classical: CD14^highCD16^neg/low; intermediate: CD14^highCD16^high; non-classical: CD14^lowCD16^high. A minimum of 10,000 events were collected, and relative expression of antigens was expressed as mean fluorescence intensity (MFI).

The following anti-human antibodies were used for staining: CD68-FITC, CD14-PercP-Cy5.5, HLA-DR-APC, CD15-PE-Cy7, CD11b-PErcP-Cy5.5, CD163-BV421, CD14-APC, HLA-DR-PE, CD33-BV421 and PD-L1-BV421 purchased from Biolegend (San Diego, CA), CD11b-PE, CD3-Alexa Fluor 700, CD19-Alexa Fluor 700, CD19-Alexa Fluor 700, CD163-Alexa Fluor 647, CD16-PE-Cy7, CD3-PE, CD19-PE and CD56-PE purchased from BD Biosciences (San Jose, CA), CD14-PE-TR and CD16 PE-TR purchased from Life Technologies (Carlsbad, CA) and CD86-FITC purchased from R&D systems (Minneapolis, MN). CD32-a-FITC Ab was purchased from Stemcell technologies, (Vancouver, Canada). CD-32-B (F-4) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, and secondary Ab anti-mouse F(ab’)2 was purchased from Thermo fisher scientific (MA, USA).
Intracellular staining of CD68 was performed as follows: PBMC were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).