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Rhod-4™, AM
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-2 is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Rhod-4™ has been developed to improve Rhod-2 cell loading and calcium response while maintaining the spectral wavelength of Rhod-2. In CHO and HEK cells Rhod-4™ AM has cellular calcium response that is 10 times more sensitive than Rhod-2 AM. AAT Bioquest offers versatile packing sizes of Quest Rhod-4 to meet your special needs, e.g., 1 mg; 10x50 µg; 20x50 µg; HTS packages with no additional packaging charge.
ATP-stimulated calcium responses of endogenous P2Y receptors were measured in CHO-K1 cells with Rhod-4™ AM (Cat# 21120) and Rhod-2 AM (Cat# 21064). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were incubated with 100 µL of dye loading solution using Rhod-4™ AM (4 µM, A and B) or Rhod-2 AM (4 µM, C and D) for 1 hour in a 37 °C, 5% CO2 incubator. The staining solution was replaced with 200 µL HHBS, then the cells were imaged before (A and C) and after (B and D) ATP treatment with a fluorescence microscope (Olympus IX71) using TRITC channel.
ATP-stimulated calcium responses of endogenous P2Y receptors were measured in CHO-K1 cells with Rhod-4™ AM (Cat# 21120) and Rhod-2 AM (Cat# 21064). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were incubated with 100 µL of dye loading solution using Rhod-4™ AM (4 µM, A and B) or Rhod-2 AM (4 µM, C and D) for 1 hour in a 37 °C, 5% CO2 incubator. The staining solution was replaced with 200 µL HHBS, then the cells were imaged before (A and C) and after (B and D) ATP treatment with a fluorescence microscope (Olympus IX71) using TRITC channel.
protocol
Protocol
CatalogSize
Price
Quantity
211201 mg
Price
211215x50 ug
Price
2112210x50 ug
Price
2112320x50 ug
Price
 
Physical properties
Dissociation constant (Kd, nM)451
Molecular weight1015.96
SolventDMSO
Spectral properties
Excitation (nm)523
Emission (nm)551
Quantum yield
0.1
1
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Instrument settings
Fluorescence microscope
ExcitationTRITC filter set
EmissionTRITC filter set
Recommended plateBlack wall/clear bottom
Fluorescence microplate reader
Excitation540
Emission590
Cutoff570
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling
Documents
Protocol
Safety Data Sheet (Catalog 21120)
Safety Data Sheet (Catalog 21121)
Safety Data Sheet (Catalog 21122)
Safety Data Sheet (Catalog 21123)
Certificate of Origin
Certificate of Analysis
Brochure: Calcium Detection Probes & Assay Kits
Brochure: Physiological Probes & Assay Kits
Flyer: Calcium Indicators
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Page updated on October 28, 2025