Rhod 2

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Rhod-2 is a calcium indicator dye used in fluorescence microscopy. It is a rhodamine-based compound that binds to calcium ions, resulting in an increase in fluorescence emission upon binding. Rhod-2 is commonly used to measure intracellular calcium levels in living cells.

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Lab products found in correlation

Fluo 4, by Thermo Fisher Scientific (13 mentions) Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (3 mentions) Mitotracker green, by Thermo Fisher Scientific (3 mentions) Mitosox, by Thermo Fisher Scientific (3 mentions) Software suite, by Nikon (3 mentions) Texas red filter cube, by Nikon (3 mentions) Fv3000, by Olympus (2 mentions) Quinacrine dihydrochloride, by Merck Group (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Mitosox red, by Thermo Fisher Scientific (2 mentions) Rhod 2, by Zeiss (2 mentions) Poly l lysine (pll), by Merck Group (2 mentions) Rhod 2, by Leica (2 mentions) Mitosox, by Leica (2 mentions) Ruthenium red, by Merck Group (2 mentions) Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions) Pluronic f 127 20 solution dmso, by Merck Group (2 mentions) Blebbistatin, by Merck Group (1 mentions) Tyrode s solution, by Thermo Fisher Scientific (1 mentions) Dcfh da, by Beyotime (1 mentions)

Close spelling variants of this product

Rhod-2 AM (225 citations) Rhod-2 acetoxymethyl ester (Rhod-2 AM), (4 citations) Rhod-2 AM probe (2 citations) Rhod 2-acetoxymethyl ester (2 citations) Rhod-2 tripotassium salt (2 citations) Rhod-2-AM Ca2+-binding dye (2 citations) Rhod-2 dye (1 citations)

66 protocols using rhod 2

Calcium Imaging of Cardiac Tissues

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A concentrated solution of Rhod-2 (Thermo Fisher Scientific) was prepared by dissolving 50 μg of the calcium indicator in 100 μl of DMSO. The solution was then diluted 1:250 in Tyrode’s solution (Alfa Aesar). Blebbistatin (Sigma-Aldrich) was added at a final concentration of 5 μM (from a 5 mM stock solution). Cardiac OBB tissues were first rinsed with Tyrode’s solution (Alfa Aesar) and incubated with the calcium indicator solution for 30 min. Next, the tissues were rinsed once with a Blebbistatin-containing Tyrode’s solution. The samples were subjected to a 555-nm light illumination, and the wide-field signal was captured by a scientific complementary metal-oxide semiconductor camera (sCMOS, Andor technology). Data were acquired using Micro-Manager open source software. During imaging, the plate containing the cardiac tissues was placed on top of a heated pad (Stoelting).

Skylar-Scott M.A., Uzel S.G., Nam L.L., Ahrens J.H., Truby R.L., Damaraju S, & Lewis J.A. (2019). Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels. Science Advances, 5(9), eaaw2459.

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Oxidative Stress and Calcium Signaling

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Oxidant stress injury was evaluated by measuring the production of intracellular ROS and mitochondrial ROS (mtROS) by DCFH-DA (Beyotime Biotechnology) and MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen, Carlsbad, CA, USA), respectively. Cells after treatment were incubated with 10 μM DCFH-DA or 1 μM MitoSOX in fetal bovine serum (FBS)-free medium for 20–30 min at 37 °C, and washed with PBS thrice. Intracellular calcium [Ca²⁺]_i overload and mitochondrial calcium [Ca²⁺]_m levels were measured by Fluo-4 and Rhod-2 (Thermo Fisher, USA), respectively. HCMECs after different treatments were incubated with 4 μM Fluo-4 and 1 μM Rhod-2 in FBS-free medium for 30 min in the dark. Immunofluorescence intensity was observed under a confocal microscope (Olympus FV3000) and analyzed using the ImageJ software (Version 1.8, NIH).

Liu M., Li S., Yin M., Li Y., Chen J., Chen Y., Zhou Y., Li Q., Xu F., Dai C., Xia Y., Chen A., Lu D., Chen Z., Qian J, & Ge J. (2024). Pinacidil ameliorates cardiac microvascular ischemia–reperfusion injury by inhibiting chaperone-mediated autophagy of calreticulin. Basic Research in Cardiology, 119(1), 113-131.

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Quantifying Mitochondrial Calcium Levels

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The levels of mitochondrial calcium were detected with fluorescent probe Rhod 2 (Thermo Fisher Scientific) using flow cytometry. Cells were seeded into 6-well plates and were cultured for 24 h. 6c at a concentration of 20 μM was added. After treated for 6 h, cells were stained with Rhod 2. Then, cells were washed three times with PBS and the fluorescence was analyzed using flow cytometry.

Wang S., Xu X., Che D., Fan R., Gao M., Cao Y., Ge C., Feng Y., Li J., Xie S., Wang C., Dai F., Gao L, & Wang Y. (2021). Reactive Oxygen Species Mediate 6c-Induced Mitochondrial and Lysosomal Dysfunction, Autophagic Cell Death, and DNA Damage in Hepatocellular Carcinoma. International Journal of Molecular Sciences, 22(20), 10987.

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Mitochondrial Ca2+ Imaging in LPS-Stimulated HEK-Blue Cells

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HEK-Blue™-hTLR4 cells were seeded on black 96-well clear-bottom plates at a seeding density of 1.25 × 10⁴ cells per well, stimulated with 100 ng/mL LPS and submitted to the NIR light exposure protocol as described above. Following treatment, the cells were stained with 20 µM Rhod2 (ThermoFisher, Abingdon, UK) and incubated for 30 min at 37 °C, 5% CO₂, and protected from light. The plates were read at 552/581 nm em/ex (FLUOStar Optima, BMG Labtech, Ortenberg, Germany). The mitochondrial Ca²⁺ levels were expressed as a percentage of the control without LPS.

Aguida B., Chabi M.M., Baouz S., Mould R., Bell J.D., Pooam M., André S., Archambault D., Ahmad M, & Jourdan N. (2023). Near-Infrared Light Exposure Triggers ROS to Downregulate Inflammatory Cytokines Induced by SARS-CoV-2 Spike Protein in Human Cell Culture. Antioxidants, 12(10), 1824.

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Visualizing Intracellular Calcium Dynamics in Astrocytes

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The [Ca²⁺]_i responses in individual cultured astrocytes were visualized by recording changes in fluorescence of conventional Ca²⁺ indicators Fura-2 (Molecular Probes), Fluo-4, or Rhod-2 (Thermo Fisher)^{5 (link),12 (link),24 (link)}. Cells were loaded with Fura-2 (5 µM; 40 min incubation; 37 °C), Fluo-4 (10 µM; 40 min incubation; 37 °C), or Rhod-2 (10 µM; 40 min incubation; 37 °C) with the addition of pluronic F-127 (0.005%). After the incubation with the dye, cultures were washed three times prior to the experiment. Changes in [Ca²⁺]_i were monitored by an inverted Olympus microscope with ×20 oil-immersion objective. Excitation light provided by a Xenon arc lamp was passed sequentially through a monochromator at 340, 380, and 490 nm (Cairn Research); emitted fluorescence was measured at 515 nm (Fura-2) or 565 nm (Rhod-2). All the experiments were performed at 37 °C.

Sheikhbahaei S., Turovsky E.A., Hosford P.S., Hadjihambi A., Theparambil S.M., Liu B., Marina N., Teschemacher A.G., Kasparov S., Smith J.C, & Gourine A.V. (2018). Astrocytes modulate brainstem respiratory rhythm-generating circuits and determine exercise capacity. Nature Communications, 9, 370.

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Mitochondrial Calcium Imaging in HDFs

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HDFs were treated with DMSO or HCP1 for 48 h. Them, Rhod2 (Thermo Fisher Scientific, MA, USA) of DMSO stock solution (1 mM) was diluted to a final concentration of 2.5 µM in the buffered physiological medium. Addition of the non-ionic detergent Pluronic^®F-127 aided in the dispersion of the nonpolar AM ester in aqueous media (Rhod2: F127 = 1:1). HDFs are normally incubated with the AM ester for 15–60 min at 20–37 °C. Cells were washed in indicator-free medium to remove any dye that was non-specifically associated with the cell surface, and were then incubated for 12–24 h to allow complete de-esterification of intracellular AM esters and eliminate cytosolic staining, while retaining mitochondrial staining. The fluorescence was detected by a confocal laser scanning microscope 700 (Carl Zeiss, Jena, Germany).

Cui X., Hao X., Wen J., Zhang S., Zhao B, & Miao J. (2022). Grp94 Inhibitor HCP1 Inhibits Human Dermal Fibroblast Senescence. Genes, 13(9), 1651.

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Rhod-2 Calcium Flux Assay

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Cells were washed with calcium flux buffer (1x HBSS, 0.1% BSA, 25 mM HEPES pH 7.4, and 2.5 mM Probenecid). Cells were incubated with 1 μM Rhod-2 (Thermo-Fisher) in calcium flux buffer in the dark for 30 min at 37°C. After incubation, cells were washed with calcium flux buffer and incubated for an additional 5 min in calcium flux buffer without Rhod-2 at 37°C. Rhod2 was measured using a BioTek Gen5 plate reader. Following 5 mins of baseline reading, Rhod-2 loaded cells were stimulated with 10 μM ionomycin. Calcium flux measurements were taken every 30 seconds until plateau was reached (approximately 20 min).

Raines L.N., Zhao H., Wang Y., Chen H.Y., Gallart-Ayala H., Hsueh P.C., Cao W., Koh Y., Alamonte-Loya A., Liu P.S., Ivanisevic J., Lio C.W., Ho P.C, & Huang S.C. (2022). PERK is a critical metabolic hub for immunosuppressive function in macrophages. Nature immunology, 23(3), 431-445.

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Cytosolic and Mitochondrial Calcium Imaging

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The 661W cells were cultured on cover glass chambered slides (Nunc Lab-Tek; Thermo Fisher Scientific, Waltham, MA, USA) with the same medium described above. After treatment with HG or HG/melatonin for 24 hours, cells were directly loaded with 2 μM Fluo-4 (Thermo Fisher Scientific) and 2 μM rhodamine-2 (Rhod-2; Thermo Fisher Scientific) for 30 mins at 37°C for cytosolic and mitochondrial Ca²⁺ imaging [47 (link), 48 (link)]. After washing, new culture medium was added, and then fluorescent images were taken under identical settings, including the light intensity, exposure time, and magnification. The average fluorescent intensity per pixel for each image was quantified without any modification using the luminosity channel of the histogram function of Photoshop 6.0 (Adobe Systems, San Jose, CA, USA). A total of 8 to 11 cell images from each group were analyzed from 3 different sets of experiments [45 (link), 46 (link)].

Chang J.Y., Yu F., Shi L., Ko M.L, & Ko G.Y. (2019). Melatonin Affects Mitochondrial Fission/Fusion Dynamics in the Diabetic Retina. Journal of Diabetes Research, 2019, 8463125.

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Quantifying ATP-Containing Vesicles in Astrocytes

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Before the experiments, the slices were incubated with 20 μM quinacrine dihydrochloride (Sigma) added to the slicing solution for 25 minutes at 37 °C. Quinacrine is a green selective marker for ATP-containing vesicles (7 (link)) or lysosomes (42 (link)). Calcium was uncaged and imaged from single astrocytes as described above, using the red Ca²⁺ indicator Rhod-2 (50 μM, ThermoFisher) instead of Fluo-4. Images of astrocyte processes with ATP vesicles were acquired before and after Ca²⁺ uncaging with 2-photon excitation at 720 nm at the soma. Adjacent areas were also imaged: we selected ROIs located a similar distance away from the astrocyte soma but without any detectable dye-filled astrocyte processes within a 5 μm perimeter minimum. With ImageJ, a constant threshold was applied to the images and particle analysis was used to quantify the number of ATP vesicles. The mean Feret’s diameter of ATP vesicles was 0.56±0.03 μm (n=2227), similar to previous reports (43 (link)).

Lezmy J., Arancibia-Carcamo L., Quintela-Lopez T., Sherman D.L., Brophy P.J, & Attwell D. (2021). Astrocyte Ca2+-evoked ATP release regulates myelinated axon excitability and conduction speed. Science (New York, N.Y.), 374(6565), eabh2858.

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Mitochondrial Membrane Potential and Calcium Dynamics

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The mitochondrial membrane potential detection kit (C2006, Beyotime, Shanghai, China) is a rapid detection kit for mitochondrial membrane potential changes using JC-1 as a fluorescent probe. Briefly, the single-cell suspensions were stained with 0.5mL working solution of JC-1 at 37 °C for 40 minutes and analyzed using a flow cytometry (Beckman Coulter, USA). Single-cell suspensions were prepared as above. Low MMP was indicated by cells with green fluorescence (JC-1 monomers) and high MMP was indicated by cells with red fluorescence (JC-1 aggregates). Mitochondrial calcium levels were measured using Rhod-2 (R1244, Thermo Fisher, USA). Calcium in the cytoplasm was quenched using CoCl₂.20 (link) Compare the average fluorescence intensity of each group.

Xu X., Zhou B., Liu J., Ma Q., Zhang T, & Wu X. (2023). Ru360 Alleviates Postoperative Cognitive Dysfunction in Aged Mice by Inhibiting MCU-Mediated Mitochondrial Dysfunction. Neuropsychiatric Disease and Treatment, 19, 1531-1542.

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