Journal: Frontiers in Immunology

Article Title: Distinguishing Sepsis From Infection by Neutrophil Dysfunction: A Promising Role of CXCR2 Surface Level

doi: 10.3389/fimmu.2020.608696

Figure Lengend Snippet: Representative diagrams showing the flow cytometry gating strategy. This analysis was performed on IDEAS ® software version 6.2. Propriety software functions are italicized. Prior to analysis, the collected events underwent camera focusing quality assessment using Gradient RMS and, for the assay which measured CD64, CCR2, and CXCR2 surface level, dead cells elimination. The selected events were firstly gated on brightfield Area and side scatter intensity to evaluate their size and granularity, respectively (A) . Single cells were separated from cell aggregates by brightfield Area (labeled as Area_BF) and preliminarily identified as neutrophils based on their granularity. CD16 positivity was used to confirm the gated cells as neutrophils (B) . The MFI of CD64, CCR2, and CXCR2 surface receptors was measured from neutrophils; which were also gated as positive or negative population using unstained samples as negative control (C) . Using Annexin-V and PI intensity, CD16-positive cells were distinguished into double-negative (healthy) cells, Annexin V-positive/PI-negative (early apoptotic) cells and double-positive (late apoptotic and necroptotic) cells (D) . Intensity threshold and Contrast morphology for the PI-channel were employed to discriminate between late apoptotic and necroptotic cells (E) . A co-staining of MPO and cell-appendant extracellular DNA was used to identify neutrophils undergoing NETosis (F) . The area of extracellular DNA, labeled as exDNA (Area), was established by subtracting BF mask from PI-channel mask; since BF and PI-channel masks delineate cell boundaries and DNA extent, respectively. CCR2, C-C chemokine receptor 2; CD, cluster of differentiation; CXCR2, C-X-C chemokine receptor 2; DNA, Deoxyribonucleic acid; MFI, Median fluorescent intensity; MPO, myeloperoxidase; PI, propidium iodide; SSC, side scatter.

Article Snippet: Fluorescent-dye conjugated antibodies used in this study are anti-human CD45-Per-CP antibody (BD Pharmingen, NJ, US); anti-human CD16-PE-Cy™7 antibody (BD Pharmingen, NJ, US); anti-human CD64-PE antibody (BD Pharmingen, NJ, US); anti-human CCR2-Alexa Fluor ® 647 antibody (BD Pharmingen, NJ, US); anti-human CXCR2-FITC antibody (BD Pharmingen, NJ, US), Annexin V-FITC antibody (BD Pharmingen, NJ, US); Propidium iodide (PI) (BD Pharmingen, NJ, US); and anti-human myeloperoxidase (MPO)-PE antibody (Bio-Rad Laboratories, CA, US).

Techniques: Flow Cytometry, Software, Labeling, Negative Control, Staining