Computational protocol: CpG-ODN Shapes Alum Adjuvant Activity Signaling via MyD88 and IL-10

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Protocol publication

[…] Three lobules (superior, middle, and post caval) of right lungs were digested with collagenase IV (2 mg/mL) and DNAse I (1 mg/mL) (Sigma-Aldrich), at 37°C for 30 min. Cell suspension was obtained after erythrocyte lysis. Extra-cellular staining to identify neutrophils and eosinophils was done using fluorochrome-conjugated antibodies (BD, San Diego CA, USA): anti-CD45 (C363.16A), -Gr-1 (RB6-8C5), -siglecF (E50-2440), and anti-major histocompatibility complex class II (MHC-II) (107630) (Biolegend, San Diego, CA, USA). For each sample, at least 1,000,000 events were collected. Cytokine production by lung cells was determined after stimulation of cells in vitro with 10 ng/mL PMA (phorbol 12-myristate 13-acetate) and 750 ng/mL ionomicin (Sigma-Aldrich, St. Louis, MO, USA) incubated with GolgiStop (BD-Biosciences, San Diego, CA, USA) for 12 h at 37°C in RPMI-1640 medium. Cell suspensions were stained with fluorochrome-conjugated antibodies against surface markers CD45 (C363.16A), CD3 (145-2C11), and CD4 (RM4-5). Cells were permeabilized and subsequently fixed with Cytofix/Cytoperm kit with GolgiPlug (BD-Biosciences, San Diego, CA, USA). For intracellular cytokine staining, we used antibodies against IL-4, IFNγ, and IL-10 (BD, San Diego, CA, USA). Only viable and non-doublet cells were considered. Cell acquisition was performed on a FACs CANTO II instrument using FACSDiva software (BD, San Diego, CA, USA). For each sample, at least 300,000 events were collected. Pools of three to five samples from the same group were used. Data were analyzed using FlowJo software (Tree Star). The number of cytokine-producing CD4+ T cells was calculated based on the total number of viable cells (trypan blue exclusion test) recovered from digested lung lobules. FACS analysis was performed with pooled samples from three to five mice. [...] Statistical analyses were performed using GraphpadPrism (V.5; GraphPad Software, USA). One-way ANOVA followed by Tukey post-test was performed, as appropriate. Differences were considered statistically significant when p value ≤ 0.05. Data are presented as mean ± SE. […]

Pipeline specifications

Software tools FlowJo, GraphPad Prism
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus
Diseases Drug Hypersensitivity, Pneumonia
Chemicals Aluminum