Skip to main content
. 2022 Jan 24;20(1):e3001513. doi: 10.1371/journal.pbio.3001513

Fig 2. Catecholamines are produced in a time-/stimulus-dependent way.

Fig 2

(A) B cells were activated for 4 h or 24 h with anti-IgM, CpG or anti-IgM/CpG and concomitantly treated with reserpine (1 μM) or NE (10 μM) for 1 h or left untreated. As control group, nonactivated B cells were used. The MFI of NS_521 (5 μM) was measured by flow cytometry. The MFI in relation to the control group is shown (n = 6). The gating strategy and representative histograms are included. (B, C) Anti-IgM/CpG-activated B cells were cultivated for 4 h or 24 h. Nonactivated B cells were used as control group. Immunofluorescence of cultivated B cells stained with NS_521 (10 μM; green) and Dapi (blue). Images were captured at 40× magnification with a Zeiss AxioVision microscope. Scale bars indicate 20 μm (main images) and 10 μm (insets). One of 3 representative images is shown (B; n = 3). The MFI of NS_521 in B cells was measured with the AxioVision software (C; n = 34 B cells/condition). (D) 1 × 107 splenic B cells from DBA/1J mice were activated with anti-IgM/CpG (5 μg/ml / 1.25 μg/ml) for 4 h and 24 h or were left inactivated. M30 dihydrochloride (MAO inhibitor, 1 μM, Tocris, catalog number: 6067) and OR-486 (COMT inhibitor, 1 μM, Tocris, catalog number: 0483) was added to cultured B cells to reduce catecholamine degradation. After incubation, EDTA (1 mM) and sodium metabisulfite (4 mM) were supplemented to cell culture medium and B cell pellets to additionally prevent catecholamine degradation. B cells were lysed in water under ultra-sonification. Cell debris were centrifuged down at 1,500 × g for 10 min at 4°C. The concentration of dopamine, NE, and epinephrine was determined by competitive ELISAs in lysed B cells and in the supernatant of B cell cultures (n = 3–4). A medium control (supernatant group) and water control (B cells) was included in the figure. Statistical significance was determined by ordinary 1-way ANOVA followed by Dunnett multiple comparison test (A) or Student t test (C, D). n.s., not significant; *p < 0.5; **p < 0.01; ***p < 0.001; ****p < 0.0001. For underlying data, see S1 Data. ANOVA, analysis of variance; COMT, catechol-o-methyltransferase; EDTA, ethylenediaminetetraacetic acid; MAO, monoamine oxidase; MFI, median fluorescence intensity; NE, norepinephrine; NS_521, neurosensor_521.