Computational protocol: IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection

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Protocol publication

[…] Cell preparations for phenotypic characterization directly ex vivo were processed and fixed within 2 h of collection, were kept on ice during processing, and were centrifuged at 4°C. Single cell suspensions were first stained with anti‐mouse CD122 (clone 5H4, eBioscience), anti‐mouse CD25 (clone PC64.5, eBioscience), anti‐mouse CD3 (clone 500A2, BD), anti‐mouse NKp46 (clone 29A1.4, BD), anti‐mouse CD69 (clone H1.2, eBioscience), anti‐mouse TRAIL (clone N2B2, eBioscience), anti‐mouse CD107a (clone 1D4B, eBioscience), and Live/Dead Fixable Near IR Dead Cell Stain Kit (Invitrogen) for 20 min at 4°C in the dark. After washing twice with HBSS + 2% FCS, cells were either fixed using 2% paraformaldehyde in PBS or permeabilized in BD Cytofix/Cytoperm for staining of intracellular cytokines for 20 min at 4°C. Before intracellular staining with anti‐mouse IFN‐γ (clone XMG1.2, eBioscience), anti‐mouse TNF (clone MP6‐XT22, eBioscience), and anti‐mouse Granzyme B (clone 16G6, eBioscience) for 20 min at 4°C in the dark, cells were washed twice with BD Perm/Wash buffer. After staining, cells were washed twice with BD Perm/Wash buffer and thereafter fixed using 2% paraformaldehyde in PBS for 20 min at 4°C. After a final wash, cells were resuspended in PBS. Samples were acquired using a BD LSRII flow cytometer and BD FACSDiva 6.0 (BD Bioscience). Data were analyzed using FlowJo for PC (TreeStar, Ashland, OR, USA). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus, Plasmodium yoelii, Homo sapiens, Plasmodium falciparum
Diseases Infection