Computational protocol: Cerebral ischemia initiates an immediate innate immune response in neonates during cardiac surgery

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Protocol publication

[…] Blood samples were kept on ice during all handling and staining procedures. Plasma samples were analyzed by multiplex immunoassay to measure levels of IL-1β, IL-6, IL-8, IL-10, TNFα, sVCAM-1 and MCP-1 with the Bio-Plex™ suspension array system (Bio-Rad Laboratories, Hercules, CA, USA) as previously described []. Neutrophil and monocyte counts were performed by manual microscopy; hematocrit and thrombocyte counts were analyzed by an automated cell counter.As shown in Figure  , monocytes were categorized into three monocyte subpopulations (CD16-, CD16intermediate (CD16int), and CD16) as proposed by Ziegler-Heitbrock et al.[,]. For neutrophil phenotyping, fluorescently labeled monoclonal antibodies (mAb) directed against CD11b (RPE, clone 2LPM19c; Dako, Glostrup, Denmark) and CD62L (FITC, clone Dreg56; BD Biosciences, Franklin Lakes, New Jersey, USA) were used for flow cytometry. For monocyte phenotyping, CD14 (Pacific Blue, 301815 clone M5E2; BioLegend, San Diego, CA, USA), HLA-DR (PE, 347401; BD Biosciences), CD16 (APC, MHCD1605, clone 3 G8, CalTag Medsystems, Buckingham, UK) and CD62L (FITC, 555543, clone Dreg 56; BD Biosciences), The FACSCanto flow cytometer (BD Biosciences) was used to record cells and FacsDiva software (BD Biosciences), Facs Express (De Novo software, Thornhill, ON, Canada) and FlowJo (Ashland, OR, USA) software were used for analysis. Neutrophils were identified according to their specific forward- and sideward-scatter signals. Expressions of CD11b and CD62L were calculated as activational markers (with CD11b increasing, and CD62L decreasing during activation). [...] Values in graphs and text are stated as numbers (% of population) or medians (interquartile range (IQR)). Differences between arterial and venous samples were tested using Wilcoxon signed-rank test. Differences between DHCA and ACP for time points from ‘30 min reperfusion’ onward were tested by Mann–Whitney U test. IBM SPSS Statistics version 19 (IBM Corp., Armonk, NY, USA) was used for statistical calculations and Prism version 5.03 was used to depict graphs (GraphPad Software, San Diego, CA, USA). […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Brain Ischemia, Heart Arrest, Stroke