cal-520®, am  (AAT Bioquest)

Journal: bioRxiv

Article Title: Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels

doi: 10.1101/2025.07.13.664578

Figure Lengend Snippet: (A) En face murine arteries were stained with fluorescent Ca 2+ indicator (Cal-520, 5 μM) and stimulated with histones (50 µg/mL) with or without Gd 3+ (10 µM) pre-treatment-Representative, cumulative Ca 2+ prevalence images from continuous fields of view (FOV) showing surgically-opened murine endothelium at baseline followed by 5 minutes of histone stimulation (top) or after pretreatment withGd 3+ followed by 5 minutes of histone stimulation. (B) Summary of Ca 2+ activity in en face cells at baseline compared to histone stimulation. (C) Summary of Ca 2+ activity at baseline, after Gd 3+ pre-treatment, and during subsequent histone stimulation. Ca 2+ signal was quantified as percent of the FOV area with oscillating cells using a specialized standard deviation method, normalized to the ionomycin-induced maximal response. (D) Wildtype (WT, left) and ORAI triple knockout (TKO, right) HEK 293 cells were stained with Ca 2+ indicator (Fura-2) and stimulated with histones (n=1). Ionomycin (10 μM) was added at the end of each experiment as a positive control. Representative traces of fluorescence over time in response to increasing concentrations of histones (top), pretreatment with Gd 3+ (10 µM) followed by histones (50 μg/mL) (middle), and Gd 3+ (10 µM) post-treatment (bottom). Quantification of maximum peak intensity for each cell following histone titration (E), Gd 3+ pre-treatment (F), and Gd 3+ post-treatment (G).

Article Snippet: Vessels were loaded with green-fluorescent Ca 2+ indicator (5 μM Cal-520 AM, Thermo Fisher Scientific) and incubated at 37 °C for 30 minutes prior to imaging

Techniques: Staining, Activity Assay, Standard Deviation, Triple Knockout, Positive Control, Fluorescence, Titration

Journal: bioRxiv

Article Title: Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels

doi: 10.1101/2025.07.13.664578

Figure Lengend Snippet: Examples of the distinct patterns of membrane movements observed during video imaging of EC monolayers over a 60-minute time frame are presented as both microscopy images and cartoons, with accompanying video supplements. (A) Ca 2+ overload with ionomycin caused similar changes in almost all cells across the field of view. The initial response consisted of rapid membrane dye incorporation into the cytoplasm concomitant with the formation of small punctate vesicular bodies located both intracellularly and in the bathing media. Following dye incorporation, numerous filopodia were formed that elongated outwards from the cell body. These filopodia were motile and not attached to the surface of the slide (A1: Supplemental Video 5). The formation of large blebs marked the final response of cells before plasma membrane integrity was irrevocably lost (A2: Supplemental Video 6). (B) Histone exposure produced a heterogenous, mosaic pattern in which some cells respond rapidly and dramatically while others appear largely unaffected (Supplemental Video 7). Dye uptake occurred in a patchy fashion in cells, often initiating at one or more regions before spreading throughout the cell body. Extracellular vesicles appear to be both released and reabsorbed. Unique to the histone response, membrane ruffling behavior was observed at the edges of some cells, with undulating movements and expansion of the membrane border away from the cell body (B1: Supplemental Video 8) whilst other cells underwent rapid retraction of the outer membrane (rounding), leaving behind retraction fibers that remain connected to the media surface and adjacent cells (B2: Supplemental Video 9). A subset of cells showed similar end-stage membrane movements with large amounts of blebbing and dye uptake (B3: Supplemental Video 10). FOV Width = 490µm. Sub panel width = 70 – 97 µm.

Article Snippet: Vessels were loaded with green-fluorescent Ca 2+ indicator (5 μM Cal-520 AM, Thermo Fisher Scientific) and incubated at 37 °C for 30 minutes prior to imaging

Techniques: Membrane, Imaging, Microscopy, Clinical Proteomics, Produced

Journal: bioRxiv

Article Title: Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels

doi: 10.1101/2025.07.13.664578

Figure Lengend Snippet: (A) Representative Z-stacks of cultured ECs after 40-minute exposure to histones (50 μg/mL) under conditions of low (0 mM), normal (1.2 mM), or high (12 mM) Ca 2+ , or with normal Ca 2+ following Gd 3+ (10 μM) pre-treatment. (B) Representative traces of individual cell cytoplasmic fluorescence (ΔF/F0) over 40 minutes for each experimental condition, quantified using ImageJ. (C) Final fluorescence (ΔF 40min /F0) for each cell with replicates displayed separately (n=3) for each condition. Kruskal-Wallis test; P<0.05 .

Article Snippet: Vessels were loaded with green-fluorescent Ca 2+ indicator (5 μM Cal-520 AM, Thermo Fisher Scientific) and incubated at 37 °C for 30 minutes prior to imaging

Techniques: Cell Culture, Fluorescence

Journal: bioRxiv

Article Title: Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels

doi: 10.1101/2025.07.13.664578

Figure Lengend Snippet: (A) Flow cytometry of histones on ECs shows exacerbation of membrane permeabilization by low Ca 2+ (0 mM) and block by high Ca 2+ (12 mM). Representative flow cytometry histograms showing FM1-43 fluorescence (PE) following incubation of EA.hy926 cells with FM1-43 dye and 50 μg/mL histones or 10 μM ionomycin for 40 minutes in buffered saline solution (HEPES-PSS) with zero added Ca 2+ (0 mM), normal Ca 2+ levels (1.2 mM), or high Ca 2+ (12 mM). The mean fluorescent intensity (MFI) was quantified using the median fluorescence for cells exposed to 50 μg/mL histones at different Ca 2+ concentrations. Kruskal-Wallis test; P<0.05. (B) Histone H3 selectively binds to membrane bound lipids in a charge dependent manner. Representative images of lipid binding strips exposed to histone H3 alone, in the presence of Gd 3+ (30 µM), or in the presence of Ca 2+ (12 mM) (left). Schematic diagram of lipid identities, adapted from the manufacturer (Right; Echelon Biosciences, Alabama, USA). (C) Summary data for histone H3 binding to cardiolipin, Phosphatidylinositol 4-phosphate (PtdIns(4)P), phosphatidic acid (PA), phosphatidylethanolamine (PE), Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylserine (PS). phosphatidylcholine (PC), sphingomyelin, and lysophosphocholine alone, or in the presence of Gd 3+ (30 µM), or Ca 2+ (12 mM). Binding was quantified in ImageJ as Area Under the Curve (AUC). N=3 for each group. Two-Way ANOVA with Bonferroni’s Correction for Multiple Comparisons; P<0.05 .

Article Snippet: Vessels were loaded with green-fluorescent Ca 2+ indicator (5 μM Cal-520 AM, Thermo Fisher Scientific) and incubated at 37 °C for 30 minutes prior to imaging

Techniques: Flow Cytometry, Membrane, Blocking Assay, Fluorescence, Incubation, Saline, Binding Assay

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-stimulated DAG production is impaired in PLCε KO cells. ( A ) A PACAP-triggered signaling cascade regulates Ca 2+ transients in chromaffin cells. PACAP binds to its high-affinity receptor, PAC1, activating Gα s . Gα s stimulates adenylate cyclase, leading to cAMP production. Elevated cAMP activates Epac and, subsequently, PLCε. PLCε hydrolyzes PIP 2 into two key signaling molecules: IP 3 and DAG. IP 3 binds to its receptors on the endoplasmic reticulum, triggering Ca 2+ release into the cytosol. This study investigates the consequences of the DAG signaling axis ( boxed ). ( B ) Representative images obtained by TIRF imaging of WT and PLCε KO cells expressing a DAG sensor during stimulation (begins at time 0) with 500 nM PACAP. The images show changes in fluorescence intensity over time, indicating DAG production. Dotted lines indicate the cell boundaries based on bright-field images. Scale bars, 5 μ m. ( C ) The percentage change in fluorescence (%ΔF/F 0 ) versus time record of the DAG sensor in WT and PLCε KO cells under basal conditions (physiological saline solution [PSS]) and during PACAP stimulation. The graph depicts the time course of DAG production after PACAP stimulation, with bold lines representing the mean response and shaded areas representing the standard error of the mean. Data were collected from two independent experiments. Sample sizes are n = 15 (WT basal), n = 11 (WT PACAP), n = 9 (KO basal), and n = 15 (KO PACAP). ( D ) Scatterplots showing the individual maximum percent change in DAG sensor fluorescence in response to PACAP in both WT and PLCε KO cells, derived from the data shown in ( C ). Data are presented as mean ± SD. Statistical significance: ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test. Not all comparisons are shown for clarity.

Article Snippet: After that, medium containing 2 mM sodium butyrate and antibiotics was added to the cells up to a total volume of 2 mL, and the culture was continued for 48–72 h. In some cases, cells were not transfected but incubated with 1 μ M of the membrane-permeant fluorescent Ca 2+ indicators Cal-520 AM (AAT Bioquest, Pleasanton, CA, 21130) or Calbryte590 AM (Cal590, AAT Bioquest, 20701) in physiological saline solutions (PSS) (145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES [pH 7.4]) for 30 min.

Techniques: Imaging, Expressing, Fluorescence, Saline, Derivative Assay

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-stimulated Ca 2+ signals and exocytosis are inhibited by NPC 15437 in a dose-dependent manner. ( A ) Representative %ΔF/F 0 versus time trace for chromaffin cells expressing Lck-GCaMP5G and stimulated with either 500 nM PACAP alone or 500 nM PACAP + 1 μ M NPC 15437 (NPC) for 45 s. Experiments were performed on a TIRF microscope. ( B ) Time series images of cells expressing Lck-GCaMP5G during stimulation with 500 nM PACAP or 500 nM PACAP + 1 μ M NPC, in the PACAP + NPC group, NPC was also applied to the bath at least 1 min before stimulation. Scale bars, 4 μ m. ( C ) Representative images of a cell expressing NPY-pHluorin stimulated with 500 nM PACAP. The outline of the cell footprint is indicated by the white lines. Arrows show the location of individual NPY fusion events in the time series. ( D ) Maximum %ΔF/F 0 for chromaffin cells stimulated with 500 nM PACAP alone or with three different concentrations of NPC (1, 10, and 50 μ M). Sample sizes are n = 38 (PACAP), n = 24 (+1 μ M NPC), n = 28 (+10 μ M NPC), and n = 31 (+50 μ M NPC). ( E ) PACAP-stimulated Ca 2+ spike area was reduced by NPC in a dose-dependent manner. Chromaffin cells stimulated with 500 nM PACAP alone or with three different concentrations of NPC (1, 10, and 50 μ M). Sample sizes are n = 37 (PACAP), n = 24 (+1 μ M NPC), n = 28 (+10 μ M NPC), and n = 27 (+50 μ M NPC). ( F ) PACAP-stimulated exocytosis was reduced by NPC in a dose-dependent manner. Chromaffin cells stimulated with 500 nM PACAP alone or with three different concentrations of NPC (1, 10, and 50 μ M) for 45 s. Sample sizes are n = 13 (PACAP), n = 13 (+1 μ M NPC), n = 13 (+10 μ M NPC), and n = 11 (+50 μ M NPC). In all PACAP +NPC groups, NPC was also applied to the bath at least 1 min earlier. Data were collected from 3 independent experiments and are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA with Kruskal-Wallis test. Statistical significance: ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: After that, medium containing 2 mM sodium butyrate and antibiotics was added to the cells up to a total volume of 2 mL, and the culture was continued for 48–72 h. In some cases, cells were not transfected but incubated with 1 μ M of the membrane-permeant fluorescent Ca 2+ indicators Cal-520 AM (AAT Bioquest, Pleasanton, CA, 21130) or Calbryte590 AM (Cal590, AAT Bioquest, 20701) in physiological saline solutions (PSS) (145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES [pH 7.4]) for 30 min.

Techniques: Expressing, Microscopy

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-mediated enhancement of activity-dependent exocytosis requires PKC signaling. ( A ) An example trace of step depolarization-evoked changes in whole-cell capacitance in a chromaffin cell. Membrane capacitance was measured before and after each step depolarization and the corresponding Ca 2+ currents (I ca ) were time integrated to estimate the Ca 2+ charge entry. Cells were held at −90 mV and currents were evoked by 50 ms step depolarizations to 0 mV. ( B ) Cumulative changes in charge (pC) were plotted against cumulative membrane capacitance changes (fF) to assess the input-output relationship. ( C ) A comparison of the pC/ΔC m relationship between cells pretreated with or without the PKC inhibitor NPC (10 μ M) for 5 min, and subsequently treated with PACAP (500 nM). PACAP was bath applied for 1 min after a control train and maintained during the second pulse train ( n = 18). NPC was maintained during the treatment train ( n = 6). Ca 2+ sensitivity of exocytosis was enhanced by PACAP. The PACAP-mediated enhancement was abolished when the cells were pretreated with NPC ( p = 0.5). ( D ) There was no change in IRP size of PACAP-treated cells either in the absence ( p = 0.06) or presence of NPC ( p = 0.09). ( E ) There was a significant increase in the RRP measured for PACAP-treated cells that was eliminated by NPC treatment ( p = 0.2). Statistical significance: ∗∗ p < 0.01; ns, not significant.

Article Snippet: After that, medium containing 2 mM sodium butyrate and antibiotics was added to the cells up to a total volume of 2 mL, and the culture was continued for 48–72 h. In some cases, cells were not transfected but incubated with 1 μ M of the membrane-permeant fluorescent Ca 2+ indicators Cal-520 AM (AAT Bioquest, Pleasanton, CA, 21130) or Calbryte590 AM (Cal590, AAT Bioquest, 20701) in physiological saline solutions (PSS) (145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES [pH 7.4]) for 30 min.

Techniques: Activity Assay, Membrane, Comparison, Control

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PKCβ and PKCε, but not PKCμ, knockdown attenuated PACAP-evoked Ca 2+ transients in chromaffin cells. ( A ) Representative trace of %ΔF/F₀ versus time for PKCβ knockdown and scrambled shRNA-transfected cells loaded with Cal520 and stimulated with 500 nM PACAP. The bottom images illustrate the signal changes at different time points in the same cell as shown in the intensity versus time record. ( B ) Knockdown of PKCβ significantly reduced the maximum amplitude of PACAP-induced Ca 2+ transients. PKCβ knockdown cells exhibited an approximately 70% reduction in the maximum %ΔF/F₀. Data are presented as mean ± SD. ( C ) The total spike area of Ca 2+ transients was also significantly decreased in PKCβ knockdown cells compared with scrambled control cells. Chromaffin cells were loaded with 1 μ M Cal520 for 30 min after transfection with the shRNA plasmid for at least 48 h. Knockdown cells were identified by RFP expression from the shRNA vector. Data are from two independent experiments ( n = 22 scrambled, n = 16 PKCβ KD). ( D ) Representative trace of %ΔF/F₀ versus time for PKCε knockdown and scrambled shRNA-transfected cells stimulated with PACAP. The bottom images show fluorescence signal changes at different time points in the same cell as shown in the trace. ( E ) Knockdown of PKCε using shRNA significantly reduced the maximum amplitude of PACAP-induced Ca 2+ transients. PKCε knockdown cells showed an approximately 70% reduction in the maximum %ΔF/F₀ compared with scrambled shRNA control cells. Data are presented as mean ± SD. ( F ) The total spike area of Ca 2+ transients was also significantly decreased in PKCε knockdown cells compared with scrambled control cells. Chromaffin cells were cotransduced with red GECO and the shRNA plasmid. Knockdown cells were identified by GFP expression from the shRNA vector. Data are from two independent experiments ( n = 18 scrambled, n = 24 PKCε KD). ( G ) Representative trace of %ΔF/F₀ versus time for PKCμ knockdown and scrambled shRNA-transfected cells stimulated with PACAP. The bottom images illustrate fluorescence signal changes at different time points in the same cell as shown in the trace. ( H ) Knockdown of PKCμ using shRNA did not significantly affect the maximum amplitude of PACAP-induced Ca 2+ transients. Data are presented as mean ± SD. ( I ) The total spike area of Ca 2+ signals was not significantly reduced in PKCμ knockdown cells compared with scrambled control cells. Chromaffin cells were loaded with 1 μ M Cal590 for 30 min after transfection with the shRNA plasmid for at least 48 h. Knockdown cells were identified by GFP expression from the shRNA plasmid. Data are from two independent experiments ( n = 29 scrambled, n = 25 PKCμ KD). Statistical significance was determined using a two-tailed unpaired t -test. ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: After that, medium containing 2 mM sodium butyrate and antibiotics was added to the cells up to a total volume of 2 mL, and the culture was continued for 48–72 h. In some cases, cells were not transfected but incubated with 1 μ M of the membrane-permeant fluorescent Ca 2+ indicators Cal-520 AM (AAT Bioquest, Pleasanton, CA, 21130) or Calbryte590 AM (Cal590, AAT Bioquest, 20701) in physiological saline solutions (PSS) (145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES [pH 7.4]) for 30 min.

Techniques: Knockdown, shRNA, Transfection, Control, Plasmid Preparation, Expressing, Fluorescence, Two Tailed Test

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: Roles for PKC signaling in the PACAP secretory pathway. Activated PLCε hydrolyzes PIP 2 into DAG and IP3. DAG activates PKCs. Activated PKCs translocate from the cytosol to the plasma membrane, where they interact with target proteins, resulting in several downstream effects: 1) enhancement of the size of the readily releasable pool of secretory granules, 2) increased Ca 2+ sensitivity of fusion, and 3) increased Ca 2+ current at negative potentials.

Article Snippet: After that, medium containing 2 mM sodium butyrate and antibiotics was added to the cells up to a total volume of 2 mL, and the culture was continued for 48–72 h. In some cases, cells were not transfected but incubated with 1 μ M of the membrane-permeant fluorescent Ca 2+ indicators Cal-520 AM (AAT Bioquest, Pleasanton, CA, 21130) or Calbryte590 AM (Cal590, AAT Bioquest, 20701) in physiological saline solutions (PSS) (145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES [pH 7.4]) for 30 min.

Techniques: Clinical Proteomics, Membrane