Computational protocol: Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts

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Protocol publication

[…] Multiple-parameter flow cytometry data were analyzed using FlowJo (Tree Star Inc., San Carlos, CA) and SPICE software (provided by Dr. Roederer, Vaccine Research Center, NIAID, NIH, USA30). Cells were gated according to forward and side scatter plots and the frequency of single, double, triple, quadruple and quintuple cytokines producing CD4+ T-cells was evaluated using boolean combination gates. As the anti-IL-4, anti-IL-5, anti-IL-13 mAbs were conjugated with the same fluorochrome, we evaluated these cytokines as a whole, identifying them as “Th2 cytokines”. After subtracting the background values, the total cytokine production and the different cytokine subsets were expressed as frequency or percentages (proportions) of the total cytokine response. The positive CD4+ T-cell response was defined as the production of any cytokines (IFN-γ and/or IL-2 and/or TNF-α and/or Th2 cytokines and/or IL-10), with 0.03% as the detection limit corresponding to at least 30 analyzed events. Functional characterization of the cytokine-producing subsets was performed only in subjects with a positive AgB cytokine response. The FACS results were generated by LP and blindly re-evaluated by a co-author, EP. The agreement of the results was high (k = 0.9) and the discrepancies were solved by discussion. […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Echinococcus granulosus, Homo sapiens
Diseases Echinococcosis