Rhod 4 am

Manufactured by AAT Bioquest

Sourced in United States

Rhod-4 AM is a fluorescent calcium indicator dye developed by AAT Bioquest. It is designed to detect and monitor calcium levels within cells. The dye exhibits a fluorescence signal upon binding to calcium ions, allowing for the visualization and quantification of intracellular calcium dynamics.

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Lab products found in correlation

Nifedipine, by Cayman Chemical (1 mentions) Black clear bottom 96 well plates, by Greiner (1 mentions) Pluronic f12, by AAT Bioquest (1 mentions) Fmax fluorimeter, by Molecular Devices (1 mentions) Powerload, by Thermo Fisher Scientific (1 mentions) Brainphys, by STEMCELL (1 mentions) Fv1000, by Olympus (1 mentions) Pluronic f 127, by Beyotime (1 mentions)

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Rhod-4 (4 citations)

4 protocols using rhod 4 am

Screening of Antiviral Compounds against SFTSV

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Huh-7, Vero 76, and SW13 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS), as described previously [15 (link)]. The source of SFTSV (YG-1) and production of polyclonal antibodies against SFTSV N protein have been described previously [15 (link)]. Rhod-4 AM (21121), used as the calcium indicator, was obtained from AAT Bioquest (Sunnyvale, CA, USA). Loperamide (L0154) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Amantadine (21364), ivabradine (15868), naloxone (15594), and nifedipine (11106) were obtained from Cayman (Ann Arbor, MI, USA).

Urata S., Yasuda J, & Iwasaki M. (2021). Loperamide Inhibits Replication of Severe Fever with Thrombocytopenia Syndrome Virus. Viruses, 13(5), 869.

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Intracellular Calcium Mobilization Assay

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Cells were seeded in black, clear bottom 96-well plates (Greiner Bio-One, Frickenhausen, Germany) and used for adenoviral transduction as described before. Thereafter, medium was removed, and cells were loaded with 2.5 μM Rhod-4 AM (AAT Bioquest, Sunnyvale, USA) in HBSS with Ca²⁺/Mg²⁺ (PAA, Pasching, Austria) containing 20 mM Hepes and 0.001% detergent (Pluronic F12, AAT Bioquest) for 30 min at 37°C and another 30 min at room temperature in the dark. GPCR agonist-evoked mobilization of intracellular calcium was measured using the f_max Fluorimeter (Molecular Devices, Biberach, Germany). Relative fluorescence units were measured every 5 s for a period of 5 min. Fluorescence intensities were normalized against non-stimulated controls.

Arnold C., Feldner A., Pfisterer L., Hödebeck M., Troidl K., Genové G., Wieland T., Hecker M, & Korff T. (2014). RGS5 promotes arterial growth during arteriogenesis. EMBO Molecular Medicine, 6(8), 1075-1089.

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Calcium Imaging of Neuronal Activity

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The calcium KIC assay was performed on separate plates of neurons to those used for the synapse density and neurite length assay. Neurons were incubated in a calcium indicator dye solution consisting of 5 μM Rhod-4 AM (AAT Bioquest #21122; Sunnyvale, CA, USA), 1X PowerLoad (Thermo Fisher Scientific #P10020), 1 μg/mL Hoechst 33342, and 2.5 mM probenecid in phenol red-free BrainPhys (Stemcell Technologies #05791; Vancouver, BC, Canada) for 40 min at 37°C. The neurons were then rinsed with and imaged under phenol red-free BrainPhys. The calcium indicator dye solution and the imaging media did not contain DMSO or ARVs. One field of view was imaged per well, including a single image in the nuclear channel followed by a calcium movie acquired at 4 frames per second for 2 minutes.

Smith A.S., Ankam S., Farhy C., Fiengo L., Basa R.C., Gordon K.L., Martin C.T., Terskikh A.V., Jordan-Sciutto K.L., Price J.H, & McDonough P.M. (2022). High-content analysis and Kinetic Image Cytometry identify toxicity and epigenetic effects of HIV antiretrovirals on human iPSC-neurons and primary neural precursor cells. Journal of pharmacological and toxicological methods, 114, 107157.

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Measuring Intracellular Calcium Dynamics

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Intracellular Ca²⁺ was measured with Rhod-4 AM (AAT Bioquest, Cat# 21121, USA) according to the manufacturer's instructions. Cultured cells were incubated with 5 μM Rhod-4 AM for 60 min in Hanks' Buffer with 20 mM Hepes (HHBS) buffer with 0.04 % Pluronic F-127 (Beyotime, Cat# ST501-0.1g, China). Then the dye working solution was replaced with HHBS containing 1 mM probenecid to remove any excess probes. Cells were imaged using an inverted fluorescence microscope (FV1000, Olympus, Japan) by monitoring the fluorescence intensity at Ex/Em = 540/590 nm with the same exposure settings for each comparison group. To obtain a stable Ca²⁺ baseline, cells were kept in Ca²⁺-free Tyrode solution for 2 min. Then 5 μm thapsigargin (TG) (MedChemExpress, Cat# HY-13433, USA) was added to induce depletion of intracellular Ca²⁺ store. After 5 min, Ca²⁺ influx was induced by adding 1 mM Calcium chloride solution. Rhod-4 fluorescence was measured and analyzed for 10 min.

Zhang H., Feng Y., Si Y., Lu C., Wang J., Wang S., Li L., Xie W., Yue Z., Yong J., Dai S., Zhang L, & Li X. (2023). Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation. Redox Biology, 69, 102983.

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