Cd15 apc

Cells were washed to remove DTT and stained with the indicated antibodies: HLA-DR-APC (BD Bioscience, Oxford, UK), CD16-PE, CD36-PE, CD15-APC (Biolegend, San Diego, CA) and CD14-biotin (Southern Biotech, Birmingham, AL). Staining combinations were as follows: CD15, CD14/CD16, CD14/HLA-DR/CD36. Streptavidin-PE-Cy5.5 tandem conjugate (eBioscience) was used as a secondary reagent to detect biotinylated primary antibodies. Following staining, samples were immediately analysed by flow cytometry (FACScalibur, Becton Dickinson, Oxford, UK).
Neutrophils were identified as CD15⁺ events. Cells of the monocyte/macrophage lineage were identified as CD14⁺. CD15⁺ neutrophils were CD14 negative, allowing distinction of these two cell types [10] (link). CD14⁺/16⁺⁺ non-classical monocytes were identified as highly CD16 positive CD14⁺ cells. Forward scatter (FSC) and side scatter (SSC) profiles were also examined. Flow cytometry data were analysed using FlowJo (Tree Star Inc, Ashland, OR).

We coated the Fluoresbrite YG Carboxylate Microspheres, 1 µm (Polysciences, 15702-10) with 2 mg/mL human immunoglobulins (IVIg, Gammunex-C) or 2 mg/mL human serum albumin (HSA, Sigma-Aldrich) and stored them at 4°C in a DPBS-NaCl buffer with 10 mg/mL bovine serum albumin (BSA, Santa Cruz Biotechnology). We then centrifuged the microspheres and replaced the supernatant with 50% autologous serum diluted in DPBS-NaCl buffers of various salt concentration (137, 200 or 300 mM). We vortexed the microspheres for 5 minutes and washed them with 1% BSA in DPBS. We resuspended them in hypertonic sodium-rich media (137, 200 or 300 mM). The microspheres were sonicated for 10 minutes, and vortexed for 5 minutes before immediate use. We isolated the neutrophils as described in 2.2., and resuspended them in respective DPBS-NaCl buffers (137, 200 or 300 mM). We added the final microsphere suspension to the cells and incubated them for 1 hour at 37°C and 5% CO₂. We stained the cells with CD15 APC (BioLegend, 1:150) and CD16 PB (BD Pharmingen, 1:150) for 30 minutes at 4°C and quantified the signal using the Gallios Flow Cytometer (Beckman Coulter). The analysis was performed in Kaluza Analysis 2.1 software (Beckman Coulter).