Computational protocol: IL-10 production differentially influences the magnitude, quality, and protective capacity of Th1 responses depending on the vaccine platform

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Protocol publication

[…] 1.5 × 106 splenocytes or cells harvested from infected ears, as previously described (), were cultured with 2 µg/ml α-CD28 (37.51) and 20 µg/ml MML or up to 2 µg/ml (each) MML peptides (Mimotopes; 15-mers overlapping by 11 corresponding to TSA and LmSTI1) for 2 h before addition of 10 µg/ml brefeldin A (BFA; Sigma-Aldrich) for an additional 4 h. For ICS, cells were washed and stained with the LIVE/DEAD Fixable Violet Dead Cell Stain (ViViD; Invitrogen) as previously described (), followed by staining with panels of antibodies against the surface markers CD3 (145-2C11), CD4 (RM4-5), CD8 (53–6.7; BioLegend), CD45.1 (A20), CD45.2 (104), and CD25 (PC61) and the intracellular markers IFN-γ (XMG1.2), IL-2 (JES6-5H4), TNF (MP6-XT22), IL-10 (JES5-16E3; eBioscience), or Foxp3 (FJK-16s; eBioscience), using the Cytofix/Cytoperm kit or Foxp3 kit (for panels including Foxp3; eBioscience) according to the manufacturer’s instructions. All ICS reagents were purchased from BD except where noted. 250,000 live lymphocytes per sample were acquired using a modified LSR II (BD) and analyzed using FlowJo software (version 8.8.6; Tree Star, Inc.) and Pestle (version 1.6.2)/SPICE (version 4.2.3; M. Roederer, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). [...] All cytokine frequencies are reported after background subtraction of the frequency of the identically gated population of cells from the same sample stimulated without antigen. Bars in figures show the median of individual mice (dots in figures). Total spleen cell counts and total numbers of viable CD4+ T cells were not significantly different between vaccine groups within the same experiment. All comparisons (bar and pie charts) of cytokine production between vaccine groups were done in SPICE v4.2.3 using a two-tailed Student’s t test assuming unequal variances or Wilcoxin-rank test (for n ≥ 4 mice). For BM chimeras, a paired analysis (t test and Wilcoxin) was used to compare responses of BM subsets within the same mouse. For challenge data, area under the curve or lesion sizes at individual time points were compared by Student’s t test using SigmaPlot (version 8; Systat Software Inc.) or JMP v5.1 (SAS Institute Inc.). Avidity was determined by a nonlinear least-squares fit of a standard Michaelis-Menten binding curve using JMP (version 5.1). Significance was determined by comparing the EC50 (effective concentration that elicits half of the maximum response) for titrations from each individual mouse. For all comparisons, p-values <0.05 are noted. […]

Pipeline specifications

Software tools FlowJo, SPICE, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Leishmania major, unidentified adenovirus
Diseases Infection, Neoplasms