a68930 hydrochloride  (Tocris)

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: DR stimulation reduces physiological cytokine secretion and activation marker expression of monocytes from women compared to men. A , B IL8 ( A ) and MCP1 ( B ) levels in supernatant from PBMCs of women and men after 24 h in cell culture, with and without in vitro stimulation by A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via ELISA; n = 11–13 per group; normalized to unstimulated control. Basal levels are presented in Supplementary Fig. 2A and B. C , D Percentage of IL8 + ( C ) and MCP1 + ( D ) B cells, monocytes, T cells, and NK cells after 24 h in culture without stimulation measured via flow cytometry; n = 5 per subtype. E , F IL8 ( E ) and MCP1 ( F ) levels in supernatant from mixed PBMCs and CD14 + monocyte-depleted PBMCs after 24 h in culture without stimulation measured via ELISA; n = 11–13 per condition. G , H Percentage of CD69 + monocytes ( G ) and expression of HLA-DR on monocytes ( H ) from women and men after 24 h in culture of mixed PBMCs, with and without stimulation by A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via flow cytometry; n = 13–14 per group; normalized to unstimulated control. Basal levels are presented in Supplementary Fig. 2G and H. Mann–Whitney test was used for comparing unpaired data of women and men, and Wilcoxon test for comparing paired data including unstimulated vs. stimulated samples as well as mixed PBMCs vs. monocytes depleted PBMCs; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques: Activation Assay, Marker, Expressing, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Control, Flow Cytometry, MANN-WHITNEY

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: Higher DRD 1 and DRD 3 expression in B cells from men was partly regulated by estradiol and testosterone. A , B Basal expression of DRD 1 , DRD 2 , DRD 3, and DRD 4 on monocytes ( A ) and B cells ( B ) from women and men measured via flow cytometry; n = 17–19 per group. C , D DRD 1 expression on B cells from women and men after 24 h stimulation with E2 (10 –8 , 10 –9 , 10 –10 M, C) and DHT (10 –7 , 10 –8 , 10 –9 M, D) in mixed PBMC culture measured via flow cytometry and normalized to DMSO control; n = 8 per condition. E , F DRD 3 expression on B cells from women and men after 24 h stimulation with E2 (10 –8 , 10 –9 , 10 –10 M, E) and DHT (10 –7 , 10 –8 , 10 –9 M, F) in mixed PBMC culture measured via flow cytometry and normalized to DMSO control; n = 8 per condition. G , H Correlation of DRD 1 expression on B cells from women and men with basal estrogen ( G ) and testosterone ( H ) levels in plasma; n = 17–18 per group. I , J Correlation of DRD 3 expression on B cells from women and men with basal estrogen ( I ) and testosterone ( J ) levels in plasma; n = 17–18 per group. Women: orange; men: grey. Unpaired t-test was used for comparing women and men, while simple linear regression was used to analyze the correlation of DR expression with sex hormone levels. One-way ANOVA with Geisser-Greenhouse correction and Dunnett multiple comparisons test was used for statistical testing of sex hormone receptor (SHR) stimulation with three concentrations of E2 and DHT. Mann–Whitney test compared the effects of SHR stimulation between women and men at the same hormone concentrations; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques: Expressing, Flow Cytometry, Control, Clinical Proteomics, MANN-WHITNEY

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: DR stimulation increased activation of female B cells. A CD86 expression on B cells from women and men after 24 h in mixed PBMC culture, with and without stimulation by A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via flow cytometry; stimulated samples are normalized to unstimulated control; n = 13–14 per group. Basal levels are presented in Supplementary Fig. 4A. B Representative flow cytometry plots of complete and CD14 + monocyte-depleted PBMCs. C CD86 expression on B cells from women and men after 24 h in monocyte-depleted PBMC culture, with and without stimulation by A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via flow cytometry; stimulated samples are normalized to unstimulated control; n = 7–8 per group. D Gating strategy for B cell subsets (1, naïve B cells: IgD + CD27-; 2, marginal zone-like B cells: IgD + CD27 + ; switched memory B cells: IgD − CD27 + ). E CD86 expression on naïve, marginal zone-like, and switched memory B cells from women and men after stimulation of mixed PBMCs with A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) for 24 h normalized to unstimulated control measured via flow cytometry; n = 13–14 per group. Mann–Whitney test was used for testing statistical significance between unpaired data of women and men. Wilcoxon test was used for the comparison of paired data including unstimulated vs. stimulated samples; *p ≤ 0.05, ***p ≤ 0.001

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques: Activation Assay, Expressing, Flow Cytometry, Control, MANN-WHITNEY, Comparison

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: Sex-specific cytokine secretion of monocytes after DR stimulation is dependent of B cells. A Representative flow cytometry plots of mixed PBMCs and CD19 + B cell-depleted PBMCs. B , C IL8 levels in supernatant from PBMCs and B cell-depleted PBMCs from women ( B ) and men ( C ) after 24 h in culture, with and without stimulation by A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via ELISA; normalized to unstimulated control; n = 8–9 per group. D , E MCP1 levels in supernatant from PBMCs and B cell-depleted PBMCs from women ( D ) and men ( E ) after 24 h in culture, with and without stimulation by A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via ELISA; normalized to unstimulated control; n = 8 per group. F , G Diagrams illustrating the interplay between B cells and monocytes after DR stimulation, showing effects on activation markers and cytokine secretion for cells from women ( F ) and men ( G ); created in BioRender.com. Wilcoxon test was used for paired data comparisons including PBMCs vs. B cells depleted as well as unstimulated vs. stimulated samples. Mann–Whitney test was used for testing statistical significance between unpaired data of women and men; *p ≤ 0.05

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Activation Assay, MANN-WHITNEY

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: Inflammatory condition turned DR stimulation-induced characteristics of female monocytes into proinflammatory phenotype. A Percentage of MCP1 + B cells, monocytes, T cells, and NK cells after 24 h in mixed PBMC culture, with or without CpG (0.195 μM) stimulation, measured via flow cytometry; n = 12 per condition. B MCP1 levels in supernatant from mixed and monocyte-depleted PBMCs after 24 h of CpG stimulation measured via ELISA; n = 14 per condition. C MCP1 levels in supernatant from PBMCs of women and men after 24 h in culture with CpG (0.195 μM) with or without A68930 (A, 10 –7 M) or Ropinirole (R, 10 –6 M) measured via ELISA; normalized to CpG control; n = 11–13 per group. Basal levels of unstimulated and CpG stimulated samples are presented in Supplementary Fig. 5C. D-G Percentage of CD69 + monocytes ( D ) and expression of HLA-DR ( E ), CD86 ( F ) and CD38 ( G ) on monocytes from women and men after 24 h in culture of PBMCs with or without CpG (0.195 μM) stimulation and after stimulation with CpG + A68930 (A, 10 –7 M) and CpG + Ropinirole (R, 10 –6 M) measured via flow cytometry; normalized to CpG control; n = 14 per group. Basal levels of unstimulated and CpG stimulated samples are presented in Supplementary Fig. 5D-G. Wilcoxon test was used for paired data comparisons including samples of two different stimulations as well as PBMCs vs. monocytes depleted. Mann–Whitney test was used for testing statistical significance between unpaired data of women and men; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Expressing, MANN-WHITNEY

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: Proinflammatory phenotype of monocytes from women after DR stimulation is independent of B cells under inflammatory condition. A , B MCP1 levels in supernatant from mixed and B cell-depleted PBMCs from women ( A ) and men ( B ) after stimulation with CpG (0.195 μM), CpG + A68930 (A, 10 –7 M) or CpG + Ropinirole (R, 10 –6 M) measured via ELISA; normalized to CpG control; n = 7–8 per group. C , D Percentage of CD69 + monocytes from women ( C ) and men ( D ) after stimulation with CpG (0.195 μM), CpG + A68930 (A, 10 –7 M) or CpG + Ropinirole (R, 10 –6 M) measured via flow cytometry; normalized to CpG control; n = 7 per group. E – J HLA-DR ( E , F ), CD86 ( G , H ), and CD38 ( I , J ) expression on monocytes from women ( E , G , I ) and men ( F , H , J ) after stimulation with CpG (0.195 μM), CpG + A68930 (A, 10 –7 M) or CpG + Ropinirole (R, 10 –6 M) measured via flow cytometry; normalized to CpG control; n = 7 per group. Wilcoxon test was used for paired data comparisons including unstimulated vs. stimulated samples as well as PBMCs vs. B cells depleted; *p ≤ 0.05

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Flow Cytometry, Expressing

Journal: Biology of Sex Differences

Article Title: Acute stimulation of PBMCs drives switch from dopamine-induced anti- to proinflammatory phenotype of monocytes only in women

doi: 10.1186/s13293-025-00689-5

Figure Lengend Snippet: Effects of DR stimulation on female and male monocytes under physiological and acute inflammatory condition. DR stimulation dampens cytokine secretion of monocytes under physiological conditions (top), dependent on activated B cells. In men, this effect could not be observed. In acute inflammatory conditions induced by CpG (bottom), DR stimulation induces a switch into a proinflammatory phenotype of monocytes from women but not from men, independent of B cells; created in BioRender.com

Article Snippet: For D 1 -like stimulation, A68930 hydrochloride (Tocris, #1534) 10 –7 , 10 –8 or 10 –9 M was added to the cells as indicated in the respective Figure Legends.

Techniques:

Journal: Scientific Reports

Article Title: Screening inducers of neuronal BDNF gene transcription using primary cortical cell cultures from BDNF-luciferase transgenic mice

doi: 10.1038/s41598-019-48361-4

Figure Lengend Snippet: Screening activators of Bdnf transcription from a dopaminergic library. ( a ) Representative result obtained using a commercially available dopaminergic library. Each compound was added into Bdnf-Luc cortical cells at 13 DIV at a final concentration of 10, 100, or 1000 nM, and luciferase activity was measured in each well 6 h later. Arrowheads show active compounds (that increased luciferase activity by more than 2-fold). For compound names, see Supplementary Table . ( b ) Changes in Bdnf expression in the presence of dopamine D 1 agonist A68930 in primary cultures of rat cortical cells. At 13 DIV, cells were treated with A68930 for 1 h, and then total RNA was prepared for RT-PCR analysis. Means ± SEM (n = 3), *** p < 0.001 and **** p < 0.0001 vs. 0 nM (one-way ANOVA with Dunnett’s multiple comparisons test). ( c ) Effect of APV or FK506 on A68930-induced Bdnf expression in cultured rat cortical cells. APV (200 μM) or FK506 (5 μM) was added 10 min before the addition of A68930 (100 nM). Means ± SEM (n = 3), * p < 0.05 and **** p < 0.0001 vs. DMSO/vehicle, †††† p < 0.0001 vs A68930/vehicle (two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: A68930 hydrochloride, (R)-propylnorapomorphine hydrochloride, and cabergoline were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Concentration Assay, Luciferase, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture