ca 2 indicator fura 2 am  (Thermo Fisher)

Journal: Science Advances

Article Title: Adaptable thermoresponsive polymer for long-term electrical coupling in plant electrophysiology monitoring

doi: 10.1126/sciadv.ady1400

Figure Lengend Snippet: ( A ) Procedure of drought stress and recovery experiment and photographs of plants under testing. Predrought is defined as 1 day before plants display wilting symptoms. ( B ) Amplitude of light-induced potential change for plants under 9 days of escalating drought (red) benchmarked against well-watered control plants (gray), showing an increase followed by decrease upon wilting. a.u., arbitrary units. ( C ) Light-induced potential change before and after inhibition of Ca 2+ channels in the same leaf. N = 3. ( D ) Light-induced potential change before and after inhibition of ROS production in the same leaf. N = 3. ( E ) Confocal images of intracellular Ca 2+ (top) and apoplastic ROS (bottom) during drought simulation, with quantification of the mean fluorescence intensity per region of interest (ROI) shown on the right. BF, bright field. Fluo-3 AM and 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA): dyes used for visualizing Ca 2+ and ROS, respectively. Scale bar, 50 μm. CK, control plants; DR, drought-stressed plants. ( F ) Plant EP signals under the light and dark conditions with starting potential aligned for all samples on each day. N = 6. ( G ) Mean potential calculated from signals in (F) 1 hour after light on or light off, showing opposing trends with time under light and dark conditions. Dark mean potential changes before visual symptom onset on day 7. Solid lines in (C), (D), and (F) are mean values, and shadows represent SD. Box plots in (B), (E), and (G) display first quartile, median, and third quartile. Whiskers are drawn at 1.5 interquartile range distance. Means are denoted by empty squares. * P < 0.05; *** P < 0.001; **** P < 0.0001; n.s., not significant. Two sample t test.

Article Snippet: Intracellular Ca 2+ ions were detected using the fluorescent Ca 2+ indicator Fluo-3 AM (MedChemExpress, HY-D0716).

Techniques: Control, Inhibition, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Chronic Overexpression of Neuronal NRG1-III in Mice Causes Long-Term Detrimental Changes in Lower Motor Neurons, Neuromuscular Synapses and Motor Behaviour

doi: 10.3390/ijms262311421

Figure Lengend Snippet: Calcium homeostasis in MNs from mice overexpressing NRG1-III. ( A ) Ca 2+ recordings from cultured MNs (14–23 DIV) reveal increased basal Ca 2+ levels in TG compared to WT. ( B ) Representative profile of intracellular Ca 2+ dynamics in basal conditions of WT and TG cultures illustrating spontaneous activity (18 DIV). ( C , D ) Quantitative analysis of Ca 2+ transient frequency ( C ) and amplitude ( D ) in cultures (14, 15, 16, 18 and 19 DIV). Sample sizes for basal Ca 2+ measurements ranged from n = 21–230 MNs and for spontaneous activity frequency and amplitude from n = 41–49 cultures, 176–235 MNs. * p < 0.05, ** p < 0.01, **** p < 0.0001 (Student’s t -test for genotype comparisons).

Article Snippet: The cultures were loaded for 3 min at 37 °C with the Ca 2+ indicator Fura-2 acetoxymethyl ester (Fura-2 AM, 5 μM, Molecular Probes), dissolved in a perfusion solution containing (in mM): 150 NaCl, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , 20 Hepes buffer (pH 7.4), 10 glucose, and 1% bovine serum albumin.

Techniques: Cell Culture, Activity Assay

Journal: Science Advances

Article Title: Matrix mechanical remodeled carrier-free nanosystem for programmable closed-loop reversal of liver fibrosis via STING alkylation

doi: 10.1126/sciadv.adz4126

Figure Lengend Snippet: ( A ) Mechanical strength and photograph of liver tissues with/without liver fibrosis. ( B ) Three-dimensional surface plots display surface hardness uniformity of hydrogels. ( C ) The response of macrophages to changes in matrix stiffness through a cycle of ECM-cell-ECM. Mechanism mechanics induce influx of Ca 2+ , mitochondrial damage, STING pathway activation, secretion of proinflammatory factors, HSC activation, and further resulting enhancement of matrix stiffness. ( D ) Approach to model the in vitro matrix mechanical environment. ( E ) Microphotographs of intracellular Ca 2+ production of RAW 264.7 cells after treatments with various concentrations of GelMA hydrogel for 24 hours and ( F ) mean fluorescence intensity (MFI) of intracellular Ca 2+ were analyzed using ImageJ software. ( G ) Mitochondrial membrane potentials of RAW 264.7 cells treated with various concentrations of GelMA hydrogel for 24 hours and ( H ) MFI of mitochondrial membrane potentials in RAW 264.7 were analyzed using ImageJ software. ( I ) Western blot detection of STING, TBK1, IRF3, p-STING, p-TBK1, and p-IRF3 in RAW 264.7 cells subjected to various concentrations of GelMA hydrogel treatments. ( J ) ELISA assay for extracellular secretion of TNF-α, IL-6, and IFN-β by RAW 264.7 cells at 24 hours after different treatments ( n = 3). ( K ) HSCs were treated with CM from RAW 264.7 cells stimulated by various concentrations of GelMA hydrogel for 24 hours. Subsequently, total collagen was quantified using Sirius Red assay ( n = 3). ( L ) Mechanical strength of HSC-T6 cells treated with CM from RAW 264.7 cells stimulated by various concentrations of GelMA hydrogel for 24 hours. GAPDH, glyceraldehyde phosphate dehydrogenase.

Article Snippet: Subsequently, the cells were harvested and stained with a mitochondrial Ca 2+ indicator (Rhod-2 AM) (MedChemExpress) for 30 min at room temperature.

Techniques: Activation Assay, In Vitro, Fluorescence, Software, Membrane, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Human induced pluripotent stem cell–derived cardiomyocytes improve recovery from myocardial infarction in non-human primates

doi: 10.1186/s13287-025-04664-0

Figure Lengend Snippet: Characterization of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A , B Characterization of differentiated hiPSC-CMs by immunofluorescence analysis of A cardiac troponin T (cTnT) and B myosin light chain 2 (MLC2v) expression; bar = 100 μm. C Flow cytometric analysis indicating > 96% purity of cTnT-positive hiPSC-CMs. D Percentage of cTnT expression in differentiated cells (n = 10 batches). E Electrical impedance study displaying a normal field potential duration of 300–450 ms in the cell mass. F Representative regular calcium transients of hiPSC-CMs incubated with a Ca 2+ indicator (Fura-2 AM). G Patch-clamp analysis showing the presence of ventricular, atrial, and nodal action potential patterns in hiPSC-CMs. H Pie chart showing the proportions of ventricular, atrial, and nodal cells in hiPSC-CMs

Article Snippet: The calcium transients of hiPSC-CMs were measured using a Ca 2+ recording system (IonOptix, USA) with a Ca 2+ indicator (Fura-2 AM, Molecular Probes, USA) under 1-Hz electrically stimulated conditions.

Techniques: Derivative Assay, Immunofluorescence, Expressing, Incubation, Patch Clamp

Journal: Biochemistry and Biophysics Reports

Article Title: Antimicrobial peptide LL-37 increases rhinovirus-induced interferon β expression in human airway epithelial cells through a Ca 2+ -dependent mechanism

doi: 10.1016/j.bbrep.2025.102105

Figure Lengend Snippet: Stimulation with LL-37 increases intracellular Ca 2+ concentration in BEAS-2B cells. (A, B) Cells were loaded with the fluorescent Ca 2+ indicator dye Fluo-4 AM and stimulated with or without LL-37 (4 μM) in Ca 2+ -containing (2.5 mM) HEPES solution. ( A ) Fluorescence was assessed continuously every 5 min for 40 min in a fluorescence microplate reader, and the time curve plotted and normalized to background fluorescence before LL-37 or vehicle was introduced. ( B ) Fluorescence determined at the endpoint (40 min) and compared to vehicle control. The fluorescence signal was normalized to fluorescence in control cells for each of the 4 independent experiments. DMSO (0.1 %) was included as vehicle control. Values are presented as means ± SEM. ∗ represents P < 0.05 vs. vehicle control determined with Student’s two-tailed unpaired t -test for single comparisons between two groups as appropriate.

Article Snippet: Cells were loaded with the Ca 2+ -indicator Fluo-4 AM (5 μM, Invitrogen) in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)-buffered salt solution with Pluronic F-127 (0.05 %, Sigma-Aldrich) for 60 min and then carefully washed two times in HEPES solution.

Techniques: Concentration Assay, Fluorescence, Control, Two Tailed Test

Journal: Biochemistry and Biophysics Reports

Article Title: Antimicrobial peptide LL-37 increases rhinovirus-induced interferon β expression in human airway epithelial cells through a Ca 2+ -dependent mechanism

doi: 10.1016/j.bbrep.2025.102105

Figure Lengend Snippet: LL-37 potentiates RV-induced IFNβ expression in the absence but not in the presence of EGTA. (A) Effects of the endosome acidification inhibitor chloroquine on LL-37-induced IFNβ transcript expression. BEAS-2B cells were pre-incubated with LL-37 (4 μM) + chloroquine (2 μg/ml) for 1 h before RV (0.15 MOI) was included. After 1 h with RV, cells were incubated for 24 h in the continuous presence or absence of LL-37 + chloroquine. ( B ) Effects of LL-37 on IFNβ mRNA expression in the absence or presence of EGTA. BEAS-2B cells were pre-incubated with LL-37 (4 μM) in the absence or presence of 0.5 mM EGTA for 1 h before RV (0.15 MOI) was included. After 1 h with RV, cells were incubated for 24 h in the continuous presence or absence of LL-37 and EGTA. ( C ) Stimulation with the Ca 2+ ionophore A23187 (10 μM) in combination with RV for 24 h potentiates RV-stimulated IFNβ transcript expression in BEAS-2B cells. A23187 was introduced 1 h prior to RV. After 1 h with RV (0.15 MOI), cells were incubated for 24 h in the continuous presence or absence of A23187. DMSO and PBS (0.1 % for both) were included as vehicle controls. Values are presented as means ± SEM. ∗, ∗∗ and ∗∗∗∗ represent P < 0.05, P < 0.01 and P < 0.0001 determined with one-way ANOVA followed by Holm-Sidak’s post-hoc analysis for multiple comparisons as appropriate. ns = not significant.

Article Snippet: Cells were loaded with the Ca 2+ -indicator Fluo-4 AM (5 μM, Invitrogen) in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)-buffered salt solution with Pluronic F-127 (0.05 %, Sigma-Aldrich) for 60 min and then carefully washed two times in HEPES solution.

Techniques: Expressing, Incubation

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: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Activation of transient receptor potential vanilloid 3 is required for keratinocyte differentiation and epidermal barrier formation

doi: 10.4196/kjpp.24.324

Figure Lengend Snippet: (A) Representative phase contrast images (200× magnification) of keratinocytes during the differentiation process from Day 0 to Day 5 in a high-calcium (> 1.3 mM Ca 2+ ) medium. (B) Representative immunoblot results showing changes in K14, K1, LRC, TRPV3, and GAPDH expression during keratinocyte differentiation. GAPDH was used as a loading control. (C) The basal marker K14 significantly decreased over time (*p = 0.01, ***p = 0.0002, ****p < 0.0001 vs . Day 0). (D) The early differentiation marker K1 showed a significant increase (*p = 0.03, **p = 0.01 vs . Day 0). (E) The late differentiation marker LRC exhibited a significant rise (*p = 0.03, **p = 0.001 vs . Day 0). (F) TRPV3 levels markedly increased after Day 1 (**p = 0.002, ****p < 0.0001 vs . Day 0). Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. TRPV3, transient receptor potential vanilloid 3; K14, keratin 14; K1, keratin 1; LRC, loricrin.

Article Snippet: [Ca 2+ ] i measurements were performed using the fluorescent Ca 2+ indicator Fura-2 acetoxymethyl ester (Fura-2 AM; Thermo Fisher Scientific).

Techniques: Western Blot, Expressing, Control, Marker