Computational protocol: Interleukin 37 is a fundamental inhibitor of innate immunity

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Protocol publication

[…] Splenocytes (5 × 106) were washed twice with PBS containing 1% BSA and incubated with an Fc-receptor blocking antibody (anti-CD16/32, eBioscience) for 15 minutes to reduce non-specific staining. The splenocytes were then stained with anti-F4/80 (eFluor 450, clone BM8), anti-CD86 (APC, clone GL1), anti-MHC class II (Alexa Fluor 700, clone M5/114.15.2), anti-NK1-1 (PE, clone PK136), and appropriate isotype controls (eBioscience), as well as anti-CD4 (PE-Cy7, clone RM4-5), anti-CD11c (FITC, clone HL3), and appropriate isotype controls (all BD) for 30 min at 4°C. Cells were then washed twice and fixed with 1% formaldehyde.Cells were analyzed using an LSR-II flow cytometer (BD Immunocytometry Systems). Between 0.5 and 1 million events were collected. Electronic compensation was performed with antibody capture beads (BD Biosciences) stained separately with individual antibodies used in the test samples. To ensure the accuracy and precision of the measurements taken from day to day quality control was performed on the LSR-II daily using the Cytometer Setup & Tracking (CS&T) feature within BD FACSDiva software. CS&T beads (BD Biosciences) were used to determine voltage, laser delays and area scaling and to track these settings over time. A manual quality control (QC) using rainbow beads was also performed daily to verify the laser delay and area scaling determined by CS&T. The data files were analyzed using the FlowJo Software (Treestar). Splenocytes were gated by their forward and side scatter profile. CD11c+ cells were selected and expression of CD86 and MHC class II was analyzed using a bivariate dot plot with biexponential scaling. Quadrant gates were set using cells stained with isotype controls and the percentage of CD11c+ cells expressing both CD86 and MHC class II was determined. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Chemical and Drug Induced Liver Injury
Chemicals Lipopolysaccharides