Computational protocol: Development and Characterization of an Effective Food Allergy Model in Brown Norway Rats

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Protocol publication

[…] Peyer’s patches and MLN lymphocytes were stained with the following mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate, phycoerythrin or allophycocyanin: anti-TCRαβ (R73), anti-CD4 (OX-35), anti-CD8α (OX-8), anti-CD45RA (OX-33), anti-NKR-P1A (10/78), anti-CD25 (OX-39) (BD Biosciences) and anti-IgA (Abcam, Cambridge, UK). Cells were labeled with saturating concentrations of conjugated mAb in PBS containing 1% FBS and 0.09% Na3N as previously described []. Negative control staining using isotype-matched mAb was included for each sample.Analyses were performed using a FC 500 Series Flow Cytometer (Beckman Coulter, FL, USA), and data were assessed by the FlowJo v7.6.5 software (Tree Star Inc,. Ashland, OR, USA). Lymphocyte populations were defined as: B (CD45RA+CD4−), B expressing IgA (IgA+CD45RA+), T (TCRαβ+), Th (TCRαβ+CD4+), Tc (TCRαβ+CD8+) and activated Th (TCRαβ+CD4+CD25+) cells. Results are expressed as percentages of positive cells in the lymphocyte population previously selected according to their forward scatter and side scatter characteristics. [...] The software package IBM SPSS Statistics 20 (SPSS Inc., USA) was used. The Levene and the Kolmogorov-Smirnov tests were applied to assess variance equality and normal distribution, respectively. Two-way ANOVA tests were used to study the effect of group and group x time interaction. The motor activity data were analyzedby two-way ANOVA for repeated measures considering the group (FA vs. IP vs. RF group) and time as the interacting factor, followed by Bonferroni’s post hoc test. To evaluate the correlation among studied variables, Pearson’s coefficient (ρ) was applied. To analyzethe results from anti-OVA antibodies, RMCP-II, βLG and cytokine concentrations, body temperature, relative gene expression, AUC of motor activity, and lymphocyte composition, non-parametric tests (Kruskal–Wallis and Mann–Whitney U) were used due to non-variance homogeneity. Differences were considered statistically significant for p values < 0.05. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Bordetella pertussis, Rattus norvegicus
Diseases Food Hypersensitivity, Hypersensitivity