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. 2023 Oct 30;221(1):e20222178. doi: 10.1084/jem.20222178

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© 2023 Yada 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 3. — GC B cell maintenance requires STIM-mediated SOC influx. (A) Schematic of experimental workflow. CD45.1/2 wild-type mice transferred with equal number of CD45.1⁺ control B1-8^high and CD45.2⁺ Stim1/2 BKO B1-8^high B cells were immunized with NP-CGG in alum. CD45.1/2 indicates cells from mice carrying both a CD45.1 and a CD45.2 allele. On the indicated time point (day 7 or 14), the mice were sacrificed and analyzed. (B) Flow cytometry of non-GC and GC B cells in the spleen of CD45.1/2 wild-type mice transferred with an equal number of CD45.1⁺ control B1-8^high and CD45.2⁺ Stim1/2 BKO B1-8^high B cells, immunized with NP-CGG in alum for 7 or 14 days. Percentages of CD45.1⁺ control and CD45.2⁺ Stim1/2 BKO cells in non-GC and GC B cells are shown. The ratios of CD45.2⁺ Stim1/2 BKO B cells to CD45.1⁺ control B cells are shown on the right. Non-GC and GC B cells are defined as FAS⁻GL7⁻B220⁺ and FAS⁺GL7⁺B220⁺ cells, respectively. *, P < 0.05, **, P < 0.001 versus non-GC B cells (Student’s t test). (C) Flow cytometry of CD45.1⁺ control and CD45.2⁺ Stim1/2 BKO GC B cells in spleen of CD45.1/2 wild-type mice transferred with equal number of CD45.1⁺ control B1-8^high and CD45.2⁺ Stim1/2 BKO B1-8^high B cells, immunized with NP-CGG in alum for 7 days. The percentages of DZ and LZ GC B cells are shown at the bottom. DZ and LZ GC B cells are defined as CXCR4^highCD86^lowCD38^lowGL7⁺ and CXCR4^lowCD86^highCD38^lowGL7⁺ cells, respectively. NS, not significant (Student’s t test). (D) Flow cytometry of CD45.1⁺ control (red histogram) and CD45.2⁺ Stim1/2 BKO GC B cells (blue histogram) in the spleen of CD45.1/2 wild-type mice transferred with equal number of CD45.1⁺ control B1-8^high and CD45.2⁺ Stim1/2 BKO B1-8^high B cells, immunized with NP-CGG in alum for 7 days. Red and blue shaded curves indicate isotype control staining of GC B cells. Numbers adjacent to histograms indicate mean fluorescence intensity for each marker of GC B cells. (E) Flow cytometry of CD45.1⁺ control and CD45.2⁺ Stim1/2 BKO GC B cells in spleen of CD45.1/2 wild-type mice transferred with equal number of CD45.1⁺ control B1-8^high and CD45.2⁺ Stim1/2 BKO B1-8^high B cells immunized with NP-CGG in alum for 7 days. Purified CD43-negative B cells from splenocytes were incubated without or with EαGFP and NP-EαGFP for 1 h before Igκ⁻FAS⁺GL7⁺ cells were gated and analyzed. Percentages of YA-e⁺GFP⁺ B cells are shown on the right. NS, not significant (Student’s t test). (B, C, and E) Data are presented as mean ± SEM for six or seven mice. Data shown are pooled from at least three independent experiments. (D) Data are representative of two independent experiments (n = 4).