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. 2020 Oct 29;218(2):e20191393. doi: 10.1084/jem.20191393

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© 2020 Müller-Winkler et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S4. — Flow cytometric gating strategy for B cell populations in spleen and bone marrow of BAFF-depleted mice and BAFF-dependent survival of MBCs in vitro. (A) WT mice were immunized with NP-CGG in alum or alum alone on day 0, followed by anti-CD40L or isotype control antibody injections on days 55, 57, and 59. Anti-BAFF or isotype control antibody was injected on days 60 and 64. (B) Gating strategy for splenic T cells (CD138⁻CD3⁺B220⁻), FO (FO B, CD3⁻CD138⁻B220⁺Fas⁻AA4.1⁻PD-L2⁻IgM^loIgG1⁻CD23⁺NP⁻), MZ (MZ B, CD3⁻CD138⁻B220⁺Fas⁻AA4.1⁻PD-L2⁻IgM^hiIgG1⁻CD23⁻NP⁻), NP⁺ IgM⁺ MBCs (IgM⁺ MBC, CD3⁻CD138⁻B220⁺Fas⁻AA4.1⁻PD-L2⁺IgM⁺IgG1⁻), NP⁻ and NP⁺ IgG1⁺ MBCs (IgG1⁺ MBC, CD3⁻CD138⁻B220⁺Fas⁻AA4.1⁻PD-L2⁺IgM⁻IgG1⁺), bone marrow mature B cells (CD138⁻B220^hi), PCs (CD138^hiB220⁻), NP⁺ IgM⁺ MBCs (CD138⁻B220^hiPD-L2⁺IgM⁺IgG1⁻), NP⁻, and NP⁺ IgG1⁺ MBCs (CD138⁻B220^hiPD-L2⁺IgM⁻IgG1⁺), blood T cells (CD3⁺B220⁻), total B cells (CD3⁻CD138⁻B220⁺), and IgM⁺, IgG1⁺, and IgG2b⁺ MBCs (CD3⁻CD138⁻B220⁺PD-L2⁺) in mice immunized with NP-CGG in alum treated as described in A 21 d after last injection of isotype control antibody. (C) WT mice were infected i.n. with X31 influenza or given PBS as a control on day 0, followed by anti-CD40L or isotype control antibody injections on days 52, 54, and 56. Anti-BAFF or isotype control antibody was injected on days 57 and 61. (D) Gating strategy for pulmonary T cells (CD3⁺CD19⁻), total B cells (CD3⁻CD19⁺), HA⁺ IgM⁺ MBCs, and HA⁺ and HA⁻ IgG1⁺, IgG2b⁺, and IgG2c⁺/IgA⁺ MBCs (CD3⁻CD138⁻CD19⁺PD-L2⁺) in lungs of mice infected with X31 influenza and then injected twice with isotype control antibody as described in C and analyzed 19 d after the last injection. (E) WT mice were immunized with NP-CGG in alum, and spleens were harvested 35 d later. (F) Flow cytometric analysis of WT splenocytes, immunized with NP-CGG in alum as described in E and cultured in 0 ng/ml or 25 ng/ml BAFF for 3 d (d3), showing gating strategy for total B cells (CD3⁻CD138⁻B220⁺), IgG1⁺, IgG2b⁺, and IgM⁺ MBCs (CD3⁻CD138⁻B220⁺PD-L2⁺). (G) Mean (±SEM) percent survival of total B cells and IgG1⁺, IgG2b⁺, and IgM⁺ MBCs at days 3 (d3; n = 12) and 5 (d5; n = 12) normalized to total cell numbers at day 0, cultured in BAFF at the indicated concentrations. Data pooled from three independent experiments (G). Mann-Whitney test was used for statistical analysis. Red boxes indicate gates used to identify cell populations.