Computational protocol: Human Cytomegalovirus Infection Enhances NK Cell Activity In Vitro

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Protocol publication

[…] The following monoclonal antibodies were used according to the manufacturer's instructions: anti-CD3 Brilliant Violet 785 (clone OKT3; Biolegend, San Diego, CA); anti-CD14 Brilliant Violet 785 (clone M5E2; Biolegend); anti-CD19 Brilliant Violet 785 (clone HIB19; Biolegend); anti-CD56 Brilliant Violet 605 (clone HCD56, Biolegend); anti-CD57 eF450 (clone TB01; eBioscience, San Diego, CA); anti-NKG2A APC (clone Z199; Beckman Coulter, Brea, CA), anti-NKG2C AF700 (clone 134591; R&D Systems, Minneapolis, MN); anti-KIR2DL1 FITC (clone 143211; R&D Systems), anti-KIR2DL1/S1 PE-Cy7 (clone EB6B; Beckman Coulter), biotinylated anti-KIR3DL1 (clone DX9; Biolegend), anti-KIR3DL1/S1 PE (clone Z27.3.7; Beckman Coulter), anti-KIR2DL2/L3/S2 PE-Cy5.5 (clone GL183; Beckman Coulter), anti-KIR2DL3/S2 FITC (clone 1F12, kindly provided by Etablissement Français du Sang, Nantes, not commercially available); anti-KIR2DL3 PE (clone 180701; R&D Systems); anti-KIR2DS4 PE-Vio770 (clone JJC11.6; Miltenyi Biotec, Bergisch-Gladbach), anti-IFNγ FITC (clone B27; BD Biosciences, Franklin Lakes, NJ); anti-CD107a PE (clone H4A3; BD Biosciences); 4′,6-diamidin-2-phenylindol and streptavidin PerCP-Cy5.5 (Biolegend).Human FcR-blocking reagent (Miltenyi Biotec) improved staining specificity. Unstained controls were used to adjust for background fluorescence. Compensation controls were performed on PBMC. The viability dye 4′,6-diamidin-2-phenylindol was used to gate out dead cells.Intracellular staining was performed after cell permeabilization using fixation buffer and permeabilization wash buffer (both Biolegend), according to the manufacturer's protocol. Samples were acquired with a BD LSR Fortessa 18-color flow cytometer (BD Bioscience). Results were analyzed using FlowJo version 10 software (Tree Star, Ashland, OR). All analyses of NK cell subpopulations were performed on PBMC. Natural killer cells were defined as the CD3-CD14-CD19-CD56+ population. Phenotypical analyses of NKG2C, CD57 and different KIRs after CMV infection were performed on a mature subset of CD56dimNKG2A− NK cells (see Figure for gating strategy). Additionally, we analyzed phenotypical and functional changes on CD56dimNKG2A-NKG2C + CD57+ cells, since these cells have been described to represent a CMV imprinted NK cell subset. [...] Phenotypic parameters as well as degranulation and IFNγ expression in total NK cells and NK cell subsets were compared between groups by generalized linear model analysis (IBM SPSS Statistics). All P values presented are 2-sided and considered significant if less than 0.05. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Human betaherpesvirus 5, Cucumber mosaic virus, Homo sapiens
Diseases Cytomegalovirus Infections, Kidney Diseases