superfusion system  (AutoMate Scientific Inc)

Journal: Neurochemical Research

Article Title: Regulation by Trace Amine-Associated Receptor 1 (TAAR1) of Dopaminergic-GABAergic Interaction in the Striatum: Effects of the Enhancer Drug (-)BPAP

doi: 10.1007/s11064-025-04337-7

Figure Lengend Snippet: Concentration-dependent effects of (-)BPAP on resting and electrical stimulation-induced [ 3 H]dopamine release from rat striatum. The resting and electrical stimulation-induced [ 3 H]dopamine release was determined as a fractional rate. (-)BPAP was added to the superfusion buffer from fraction 8 in a concentration range from 10 –15 to 10 –5 mol/L. A : Resting [ 3 H]dopamine release was determined in fractions 3 (basal outflow in the absence of drug, B1) and 17 (basal outflow in the presence of drug, B2) and the B2/B1 ratio was calculated. The B2/B1 value was 0.95 ± 0.05 (n = 8) in control c experiments. (-)BPAP added in these concentrations was without effect on resting [ 3 H]dopamine release. One-way ANOVA followed by the Dunnett’s test, F(11,40) = 0.464, p = 0.914, mean ± S.E.M., n = 3–8. B : The effect of (-)BPAP on electrical stimulation-induced [ 3 H]dopamine release. Electrical stimulation (40 V, 10 Hz, 2-ms for 3 min) was applied in the 1st (absence of drug, S1) and 2nd (presence of drug, S2), stimulations carried out in fractions 4 and 18 and the release was expressed as S2/S1 ratio. The S2/S1 value was 0.77 ± 0.05 (n = 8) in control experiments. Note: (-)BPAP exerted a dual-effect: it increased electrical stimulation-induced [ 3 H]dopamine release in 10 –12 , 10 –11 and 10 –6 , 10 –5 mol/L concentrations. One-way ANOVA followed by the Dunnett’s test, F(11,40) = 7.743, p = 0.0001, *p < 0.05, mean ± S.E.M., n = 3–8

Article Snippet: After loading the tissues with [ 3 H]dopamine, the slices were transferred into low volume (0.3 ml) superfusion chambers (Experimetria Kft, Budapest, Hungary) and superfused with aerated and preheated Krebs-bicarbonate buffer.

Techniques: Concentration Assay, Control

Journal: Neurochemical Research

Article Title: Regulation by Trace Amine-Associated Receptor 1 (TAAR1) of Dopaminergic-GABAergic Interaction in the Striatum: Effects of the Enhancer Drug (-)BPAP

doi: 10.1007/s11064-025-04337-7

Figure Lengend Snippet: The effect of (-)BPAP on resting and electrical stimulation-induced [ 3 H]GABA release from rat striatum. The resting and the electrical stimulation-induced [ 3 H]GABA release was determined as a fractional rate. (-)BPAP was added in a concentration range from 10 –15 to 10 –5 mol/L to the superfusion buffer from fraction 8 and maintained through the experiment. A : The B2/B1 ratio indicates the effect of (-)BPAP on resting [ 3 H]GABA release determined in fractions 4 (absence of drug, B1) and 14 (presence of drug, B2). The B2/B1 value was 0.87 ± 0.04 (n = 10) in control experiments (c). One-way ANOVA followed by the Dunnett’s test, F(11,58) = 2.136, p = 0.031, the Dunnett’s test did not indicate significant changes, mean ± S.E.M., n = 4–10. B : The effect of (-)BPAP on electrical stimulation-induced [ 3 H]GABA release. The stimulation (40 V, 20 Hz, 2-ms for 6 min) was applied in the presence and absence of the drug in collected fractions 15 and 16. Control (c) [ 3 H]GABA release was 2.02 ± 0.34 per cent of content released (n = 10). Note: (-)BPAP exerted a dual-effect: it increased electrical stimulation-induced [ 3 H]GABA release in 10 –13 and 10 –12 and 10 –9 to 10 –7 mol/L concentrations. Data in Fig. 3B were subjected to logarithmic transformation and one-way ANOVA followed by the Dunnett’s test, F(11,58) = 2.825, p = 0.0051, *p < 0.05, mean ± S.E.M., n = 4–10

Article Snippet: After loading the tissues with [ 3 H]dopamine, the slices were transferred into low volume (0.3 ml) superfusion chambers (Experimetria Kft, Budapest, Hungary) and superfused with aerated and preheated Krebs-bicarbonate buffer.

Techniques: Concentration Assay, Control, Transformation Assay

Journal: Nature Communications

Article Title: Single-cell analysis identifies the CNP/GC-B/cGMP axis as marker and regulator of modulated VSMCs in atherosclerosis

doi: 10.1038/s41467-024-55687-9

Figure Lengend Snippet: a Western blot analysis of VSMC lysates. Applied antibodies and expected molecular weights of the target proteins are indicated. One representative blot out of three independent experiments is shown. b cGMP/FRET imaging of primary VSMCs isolated from global cGMP sensor mice. VSMCs were stimulated with (left) ANP, (middle) CNP, or (right) DEA/NO (80 nM each, red bars). Cyan and yellow traces show CFP and YFP fluorescence of the sensor, respectively. Black traces indicate the intracellular cGMP concentration over time (ratio trace of CFP/YFP or R ~ [cGMP]); shown are means of all cells that reacted to the respective stimulus (left: 20 analyzed out of 26 recorded cells; middle: 33 out of 35 cells; right: 42 out of 57 cells; one coverslip each from the same cell isolation). The scale bars indicate the time and percent change of the traces relative to baseline. c Representative cGMP/FRET measurements (ratio traces R ~ [cGMP]) of individual VSMCs that were consecutively stimulated with ANP, CNP, and DEA/NO (red bars, concentrations indicated in the panel). Based on the analysis of 438 out of 516 recorded cells (on three coverslips from one cell isolation), the cells were classified as “ANP-preferring” (orange, 124 cells), cells without a clear preference for ANP or CNP (“ANP ~ CNP”, green, 65 cells), and “CNP-preferring” (cyan, 249 cells). The black scale bars indicate the time and percent change of the traces relative to baseline. The pictures on top show the cGMP sensor expressing VSMCs visualized by the YFP fluorescence of cGi500. In the right picture, the cGMP response pattern of each cell is highlighted using the same color code as for the cGMP traces in the lower panel. White scale bars, 50 μm. d Comparison of cGMP responses in VSMCs from control and global GC‑B knockout mice. cGMP/FRET measurements (ratio traces R ~ [cGMP]) were performed as in ( c ). The responses are representative of “ANP-preferring” (orange) and “CNP-preferring” (cyan) cells. Shown are means from one representative measurement per genotype (from top to bottom: 55 cells, 68 cells, 6 cells, 25 cells). For the control measurement, ratio traces for 61 out of 87 recorded cells (on one of two coverslips from one cell isolation) are shown. Cells classified as ANP ~ CNP cells are not shown and, therefore, not included in the total count of recorded cells. For the GC-B knockout measurement, ratio traces for 93 out of 137 recorded cells (on one of two coverslips from one cell isolation) are shown. As the control measurement lasted longer, some data points from the baseline were omitted (gaps in the control traces) to align the drug applications without distorting the time axis. The scale bars indicate the time and percent change of the traces relative to baseline. These results were confirmed by an independent experiment with GC-B knockout VSMCs that were transfected with the cGi500 biosensor. As detailed in the Methods section, cells showing a poor quality of their ratio traces were excluded from analysis. The respective numbers of analyzed cells out of total recorded cells are indicated above. Source data including exact p-values and applied statistical tests are provided in the file.

Article Snippet: The superfusion system consisted of an FPLC pump (Pharmacia P-500, GE Healthcare), FPLC injection valves (Pharmacia V-7, GE Healthcare), a vacuum pump (Laboport N86, KNF Neuberger), a 2 mL sample loop, tubing (Tygon S3 E-3603, Saint-Gobain), and a superfusion chamber for 12 mm coverslips (RC-25, #64-0232, Warner Instruments) attached to a magnetic chamber holder (PM-1, #64-1526, Warner Instruments).

Techniques: Western Blot, Imaging, Isolation, Fluorescence, Concentration Assay, Cell Isolation, Expressing, Comparison, Control, Knock-Out, Transfection

Journal: Nature Communications

Article Title: Single-cell analysis identifies the CNP/GC-B/cGMP axis as marker and regulator of modulated VSMCs in atherosclerosis

doi: 10.1038/s41467-024-55687-9

Figure Lengend Snippet: To correlate the phenotype and cGMP response pattern of an individual cell, we have used a mapping method based on gridded coverslips. For details, see Methods section and Supplementary Fig. . Primary VSMCs from global cGMP sensor mice were grown and imaged for cGMP as described in Fig. , followed by immunofluorescence staining for marker proteins. The panels show representative images and the quantitative evaluation of fluorescence intensities (normalized to the mean of ANP-preferring cells) of individual VSMCs stained for a , b contractile markers αSMA and SM22α, c , d αSMA and the non-SMC protein S100A4 and e , f the non-SMC protein PDGFRα. ANP-preferring cells are indicated by orange cell borders and data points, and CNP-preferring cells by cyan cell borders and data points. Each data point represents an individual VSMC. Data are shown as mean + SEM. b 112 cells were analyzed out of 114 recorded cells on two coverslips from one cell isolation. d 105 cells were analyzed out of 113 recorded cells on four coverslips from one cell isolation. f 97 cells were analyzed out of 115 recorded cells on five coverslips from one cell isolation. Statistical significance is indicated by asterisks (*** p < 0.001). Scale bars, 50 μm. DNA was stained with Hoechst No. 33258 (blue). Similar results were obtained in another independent experiment. As detailed in the Methods section, cells that showed a poor quality of their ratio traces or that could not be analyzed in the immunostaining due to limitations of the mapping technique were excluded from analysis. The respective numbers of analyzed cells out of total recorded cells are indicated above. Cells classified as ANP ~ CNP cells are not shown and, therefore, not included in the total count of recorded cells. Source data including exact p-values and applied statistical tests are provided in the file.

Article Snippet: The superfusion system consisted of an FPLC pump (Pharmacia P-500, GE Healthcare), FPLC injection valves (Pharmacia V-7, GE Healthcare), a vacuum pump (Laboport N86, KNF Neuberger), a 2 mL sample loop, tubing (Tygon S3 E-3603, Saint-Gobain), and a superfusion chamber for 12 mm coverslips (RC-25, #64-0232, Warner Instruments) attached to a magnetic chamber holder (PM-1, #64-1526, Warner Instruments).

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Cell Isolation, Immunostaining

Journal: Nature Communications

Article Title: Single-cell analysis identifies the CNP/GC-B/cGMP axis as marker and regulator of modulated VSMCs in atherosclerosis

doi: 10.1038/s41467-024-55687-9

Figure Lengend Snippet: a Working principle of the GC-B LacZ reporter gene. X-gal staining of cells from GC-B LacZ reporter mice leads to blue stained nuclei in cells that express the GC-B gene at the time of staining. Note that GC-B-dependent cGMP signaling in heterozygous GC-B LacZ reporter cells is ensured by the presence of the GC-B wildtype allele. Cyan handles in the nucleus indicate indigo dye that stains nuclei of GC-B expressing cells. b Development of GC‑B expression in primary VSMCs grown for 4–7 days in the presence of 10% FCS. Cells with presumably high GC‑B expression were identified by their X‑gal-stained nuclei (X-gal + /GC-B high cells). Each data point represents the percentage of X-gal + /GC-B high cells in a random field of view. Data are shown as mean + SEM ( n = 10 regions on two coverslips per time point from one cell isolation). Statistical significance vs. day 7 is indicated by asterisks (** p < 0.01; *** p < 0.001). Similar results were obtained in another independent experiment. The inset shows a representative region of cultured VSMCs at day 7. White arrowheads indicate the position of X‑gal+ nuclei. DNA was stained with Hoechst No. 33258 (blue). Note that nuclei showing an intense X-gal staining (dark blue) were not visible in the Hoechst No. 33258 channel due to interference of the X-gal precipitate with Hoechst fluorescence. Scale bars, 100 µm. c , Effect of serum concentration on ANP/CNP preference of primary VSMCs. Cells were grown under standard conditions in cell culture medium supplemented with 0.1%, 10% or 20% FCS for 48 h before cGMP/FRET measurements. Drugs were applied as shown in Fig. . Bars indicate the fraction of cells in each category under each condition (0.1% FCS: 167 cells were analyzed out of 172 recorded cells on three coverslips from three cell isolations; 10% FCS: 96 cells were analyzed out of 98 recorded cells on two coverslips from two cell isolations; 20% FCS: 121 cells were analyzed out of 123 recorded cells on two coverslips from two cell isolations). d Effect of passaging on ANP/CNP preference of VSMCs. Primary and passaged VSMCs were grown under standard conditions in 10% FCS and analyzed by cGMP/FRET measurements. Bars indicate the fraction of cells in each category under each condition (p0: 284 cells were analyzed out of 291 recorded cells on five coverslips from two cell isolations; p2: 214 cells were analyzed out of 232 recorded cells on six coverslips from two cell isolations; p4: 123 cells were analyzed out of 167 recorded cells on five coverslips from two cell isolations). On the right, representative images of the VSMCs (visualized by the YFP fluorescence of cGi500) used for cGMP/FRET measurements are shown. Scale bars, 100 μm. In panels c and d, statistically significant differences in the distribution of ANP-preferring, ANP ~ CNP, and CNP-preferring cells under different conditions are indicated by asterisks (* p < 0.05; *** p < 0.001). As detailed in the Methods section, cells showing a poor quality of their ratio traces were excluded from analysis. The respective numbers of analyzed cells out of total recorded cells are indicated above. e Effect of passaging on GC-B expression in VSMCs detected by Western blotting. Applied antibodies and expected molecular weights of the target proteins are indicated on the left. Similar results were obtained in another independent experiment, where low passages (p1-p3) of VSMCs were compared. Source data including exact p-values and applied statistical tests are provided in the file.

Article Snippet: The superfusion system consisted of an FPLC pump (Pharmacia P-500, GE Healthcare), FPLC injection valves (Pharmacia V-7, GE Healthcare), a vacuum pump (Laboport N86, KNF Neuberger), a 2 mL sample loop, tubing (Tygon S3 E-3603, Saint-Gobain), and a superfusion chamber for 12 mm coverslips (RC-25, #64-0232, Warner Instruments) attached to a magnetic chamber holder (PM-1, #64-1526, Warner Instruments).

Techniques: Staining, Expressing, Cell Isolation, Cell Culture, Fluorescence, Concentration Assay, Passaging, Western Blot