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. 2020 Aug 12;11:1946. doi: 10.3389/fimmu.2020.01946

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Copyright © 2020 Taylor, Brewer, Nedeljkovic-Kurepa, Yang, Reddy, Abraham, Barnes and Deutschman.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effects of immune education on B cell populations 24 h post-CLP. C57Bl/6 laboratory mice underwent education or treatment with isotype control antibody. Thirty-five days later mice were euthanized (T₀) or subjected to CLP. Mice subjected to CLP were euthanized 24 h later. Spleens were homogenized and B cell populations were analyzed using flow cytometry. Each point represents results in an individual animal, central horizontal line indicates mean, upright and inverted Ts indicate standard deviation, data representative of two independent experiments. Filled circles – T₀ in control mice; Filled square – T₀ in immune educated mice; Open circle – 24 h post -CLP in control mice; Open square – 24 h post-CLP in immune educated mice. Data analyzed using two-way ANOVA with Sidak’s post hoc correction for multiple comparisons. *p < 0.05, significantly different from value in control mice at same time point; ^#p < 0.05, slope of line connecting mean at T₀ to mean value 24 h post-CLP significantly different than same line in control mice. (A) Total splenic B cells. Gating: FSC/SSC, singlets, Live, CD19⁺/B220⁺; N = 5–9/group. (B) Total follicular (Left) and marginal zone (Right) B cells per spleen. Gating: Follicular B cells: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^lo/CD21/35^lo. N = 5–6/group. (C) Total marginal zone B cells per spleen. Gating: Marginal zone: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^hi/CD21/35^hi. N = 5–6/group. (D) Total follicular I (left) and follicular II (right) B cells per spleen. Gating: Follicular I B cells: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^lo/CD21/35^lo, IgD⁺/IgM^lo. Follicular II B cells: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^lo/CD21/35^lo, IgD⁺/IgM^mid N = 5–6/group. (E) Total germinal center B cells per spleen. Gating: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^lo/CD21/35^lo, GL7⁺; N = 5–6/group. (F) Total plasma cells per spleen. Gating: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138⁺; N = 7/group. (G) Total CD69 + follicular (left) and CD69 + marginal (right) B cells per spleen. Gating: Follicular B cells: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^lo/CD21/35^lo, CD69⁺/IgM⁺. Marginal B cells: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD93^–, B220⁺/CD138^–, IgM^hi/CD21/35^hi, CD69⁺/IgM⁺ N = 3–4/group.