Intracellular Ca2+ levels were quantified using a Ca2+ quantification kit (Abcam, ab112115) following the standard manufacturer’s protocol. Fluorescence was determined using a microplate spectrophotometer at Ex/Em = 540/590 nm (Varioskan LUX, Thermo). Additionally, cytosolic Ca2+ levels were measured by flow cytometric estimation of Fluo-4 AM. The cells were collected and loaded with 5 μM Fluo-4 (Beyotime, ab145254) for 30 min at 37° C, and then resuspended in 500 μL of phosphate-buffered saline. Fluorescence signals were recorded with a flow cytometer at Ex/Em =488/525 nm and analyzed with NovoExpress software (NovoCyte, ACEA Biosciences, San Diego, CA, USA).
Fluo 4
Manufactured by Beyotime
Sourced in China
Fluo-4 is a fluorescent calcium indicator used for measuring intracellular calcium levels. It is a small molecule that undergoes a significant increase in fluorescence upon binding to calcium ions. Fluo-4 is designed to provide a reliable and sensitive method for monitoring calcium dynamics within cells.
Automatically generated - may contain errors
Lab products found in correlation
Novocyte, by Agilent Technologies (1 mentions)
Novoexpress software, by Agilent Technologies (1 mentions)
Ca2 quantification kit, by Abcam (1 mentions)
Rhodamine labeled phalloidin, by Merck Group (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
β actin 60008 1 ig, by Proteintech (1 mentions)
A16686, by ABclonal (1 mentions)
β actin ac038, by ABclonal (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fluorescence microplate reader, by Tecan (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Laser confocal microscopy, by Leica (1 mentions)
Lcs sp8 sted, by Leica (1 mentions)
Fluo 4 am, by Beyotime (1 mentions)
9 protocols using fluo 4
1
Intracellular Calcium Quantification
2
Cardiomyocyte Signaling Pathway Assay
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AsIV was obtained from Nanjin Jingzhu Biotechnology Company (purity >98% measured by HPLC; Nanjing, China). Iso(I5627), GdCl3(G7532),and 2-APB(D9754), Dimethyl Sulfoxide (DMSO) and rhodamine-labeled phalloidin were purchased from Sigma-Aldrich (St. Louis, MO, United States). NPS2143 (S2633) was purchased from Selleck Chemicals (Houston, TX, United States). Antibodies against CaMKII(AB11287), SERCA2a (AB41825), and IP3R(AB12382) were purchased from Absci (Baltimore, MD, United States). Antibodies against NFAT-3 (ab99431), GATA-4 (ab84593), Bcl-2 (ab196495), and Bax(ab5313) were purchased from Abcam (Cambridge, MA, United States). Antibodies against CaN (13198-2-AP), CaSR (19125-1-AP), and β-actin (60008-1-Ig) were purchased from Proteintech Biotechnology (Wuhan, China). Nuclear and mitochondrial extraction kits were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). A 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) kit and Fluo-4 were obtained from the Beyotime Institute of Biotechnology (Nanjing, China). A terminal deoxynu-cleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) kit was purchased from Roche (Darmstadt, Germany). TRIzol reagent, ANP and BNP primers were obtained from TaKaRa Biotechnology Co. (Dalian, China).
3
AS-IV Modulates Pulmonary Endothelial Cells
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AS‐IV was obtained from Nanjing Jingzhu Biotechnology Company (purity > 98% measured by HPLC, Nanjing, China). Human pulmonary artery endothelial cells were purchased from BLUEFBIO (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma Aldrich (St. Louis, MO, United States). CCN1‐small interfering lentiviruses were purchased from Shanghai Just Science. (Shanghai, China). Recombinant CCN1 protein was purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS, LOT 0001644044) was purchased from Sigma. EGF was purchased from purchased Sigma (St. Louis, MO, USA). The A5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐lbenzimidazol‐carbocyanine iodide (JC‐1) kit and Fluo‐4 were obtained from the Beyotime Institute of Biotechnology (Nanjing, China). Antibodies against Bcl‐2 (A0208), Bax (A0207), Caspase‐3 (A2156), CCN1 (A7632), ERK (A16686) and p‐ERK (AP0886), and β‐actin (AC038) were purchased from ABclonal (ABclonal Technology Co.,Ltd.). A terminal deoxynucleotidyl transferase‐mediated dUTP Nick‐End Labeling (TUNEL) kit was purchased from Roche (Darmstadt, Germany).
4
Two-Photon Imaging of Hippocampal Neurons
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Mouse brain was removed and then the acute transverse brain slices at 200-μm intervals were prepared beginning at approximately 10 mm from the rostral ends using the leica biosystems vibratome (Leica Microsystems, Wetzlar, Germany) in oxygenated mildly modified ACSF (95 mM NaCl, 1.8 mM KCl, 1.2 mM NaH2PO4, 7mM MgSO4, 26 mM NaHCO3, 15 M Glucose, 50.5 mM Sucrose). Next, the slices were transferred to a chamber where they were incubated in continually oxygenated ACSF containing 2 uM Fluo-4 (Beyotime Biotechnology, Shanghai, PR China) at room temperature for 30 min. Two-photon imaging of Ca2+ in hippocampal neurons was conducted using Femto3D Atlas (Femtonics, Budapest, Hungary) equipped with a 25 X water immersion objective (MRD77220, numerical aperture: 1.10, working distance: 2 mm) (Nikon, Tokyo, Japan), and a broadly tunable laser which was set at a wavelength of 920 nm and a repletion rate of 1.7 Hz.
5
Quantifying Calcium Dynamics with Fluo-4
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After 24 h of CBG treatment, cells were collected, washed, and stained with 4 μM Fluo-4 (Beyotime) staining solution at 37 °C for 30 min in the dark. Thereafter, the cells were washed with PBS and incubated for 20 min. Ca2+ activity was then detected using a fluorescence microscope (Nikon) or fluorescence microplate reader (Tecan) with excitation at 488 nm and emission at 515 nm.
6
Measuring Intracellular Calcium Dynamics
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Cardiomyocytes were cultured on Matrigel-coated confocal dishes and loaded with the calcium-sensitive fluorescent probe Fluo-4 (Beyotime, # S1061S, China). The intracellular calcium levels were monitored using a laser confocal microscopy (Leica, German) in line-scan mode. The acquired images were analyzed using Image J and Igor to detect changes in intracellular calcium levels.
7
Cellular and Mitochondrial Calcium Measurement
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The levels of cellular calcium and mitochondrial calcium were measured by Fluo-4, AM (Beyotime, China) and Rhod-2, AM (Maokang, China) according to the manufacturer’s instructions, respectively. After treatment, the cells were washed with PBS for 3 times and incubated with working solution for 30 min in the cell incubator. The results were observed by the fluorescent microscope.
8
Intracellular and Mitochondrial Calcium Imaging
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Intracellular Ca2+ and mitochondrial Ca2+ levels were measured using Fluo-4, AM (Beyotime, China) and Rhod-2, AM (Maokang, China), respectively, following the manufacturer’s instructions. After treatment, cells were washed three times with PBS and then incubated with the working solution at 37 °C for 30 min. The images were visualized using laser scanning confocal microscopy.
9
Calcium Imaging of Astrocytic TRPV4
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Fluorescent calcium imaging was performed as previously described [18 (link)]. Briefly, primary cultured astrocytes were seeded in confocal dishes and were incubated with Fluo-4 AM (3 μM, Beyotime Biotechnology, China, #S1060) for 30 min at 37 °C in the dark, and then washed with PBS three times, subsequently followed by incubation in PBS for another 30 min at 37 °C, permitting Fluo-4 AM de-esterification and binding with cytosolic Ca2+. Confocal fluorescent calcium images of astrocytes were obtained by a confocal laser scanning microscope (Leica-LCS-SP8-STED, Leica, Wetzlar, Hesse, Germany) and real-time fluorescent calcium imaging was recorded. The ΔF/F0 ratio is calculated as (F–F0)/F0. Here, F0 is the basal fluorescence intensity of Fluo-4, while F represents the real-time fluorescence intensity before and after adding GSK1016790A (TargetMol, USA, #T6848), a specific TRPV4 agonist, and ΔF is the difference between F and F0.
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