Computational protocol: Simultaneous detection of decidual Th1/Th2 and NK1/NK2 immunophenotyping in unknown recurrent miscarriage using 8-color flow cytometry with FSC/Vt extended strategy

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Protocol publication

[…] The initial gating was applied with FSC/Vt gating methods, compared with FSC/ SSC regular gating methods. Adhesion events were excluded. Live cells from the FSC/Vt gates or the Vt/histogram were gated with the CD3/CD56 quadrant gate. The CD4/CD8 quadrant gate was set within the CD3+CD56– subsets, and the CD56brightCD16– cells were identified within the CD3–CD56+ subsets. The NKT-like cells were identified as the CD3+CD56+ subsets. The quadrant gates of IFN-γ/IL-4 were set for the Th (CD3+CD4+), Tc (CD3+CD8+), dNK (CD3–CD56brightCD16−), and NKT-like lymphocytes (CD3+CD56+). Each subpopulation was quantified as a percentage of the parental population. Based on these two methods, centralised and extended scales were attempted. The initial centralised gate was set on the brightest part of a pseudocolor plot. The initial extended gate was set to include the peripheral of the brightest part. Generally, FSC/SSC (central), FSC/SSC (extended), FSC/Vt (central), and FSC/Vt (extended) gates were applied. All of the flow data were processed with FlowJo software 10.0 (TreeStar, Ashland, OR, U.S.A.). [...] The statistical analyses were performed using SPSS Statistics software 22.0 (IBM, Armonk, NY, U.S.A.). The age and cell percentage data are presented as the mean ± S.E.M. (M ± S.E.M.). A two-way ANOVA under a significance level of 0.05 for factorial design was applied to compare the effects of the four gating strategies. For two independent samples, a t test at a two-tailed significant level of 0.05 was performed for comparison of the cell percentages between the two subject groups. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens