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Rhod-2 AM

Cat. No. M10357
Rhod-2 AM Structure
Size Price Availability Quantity
1mg USD 550  USD550 In stock
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Quality Control
  • Current batch:
  • Purity >98%, Biological Stain
  • COA
  • MSDS
Biological Activity

Rhod-2 AM is a fluorescent, mitochondrial probe (λex=552 nm, λem=581 nm). Rhod-2 AM is cell-permeable version of Rhod-2. The long-wavelength Rhod-2 Ca2+ indicators are valuable alternatives to Fluo-3 for experiments in cells and tissues that have high levels of autofluorescence. 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.


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Rhod-2 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Rhod-2 AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Rhod-2 AM Working Solution
On the day of the experiment, either dissolve Rhod-2 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Rhod-2 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Rhod-2 AM.
Note If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators.

SAMPLE EXPERIMENTAL PROTOCOL

Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
  1. Prepare cells in growth medium overnight.
  2. On the next day, add 1X Rhod-2 AM working solution into your cell plate.
    Note If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
  3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
    Note Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
  4. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
  5. Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a TRITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm.


Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)
Species Mouse Rat Rabbit Guinea pig Hamster Dog
Weight (kg) 0.02 0.15 1.8 0.4 0.08 10
Body Surface Area (m2) 0.007 0.025 0.15 0.05 0.02 0.5
Km factor 3 6 12 8 5 20
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information
Molecular Weight 1123.96
Formula C52H59BrN4O
CAS Number 145037-81-6
Purity >98%, Biological Stain
Solubility DMSO 1~5mM
Storage -20°C, protect from light, dry, sealed
References

[1] Xudong Peng, et al. BMC Ophthalmol. Phospholipase Cγ2 is critical for Ca 2+ flux and cytokine production in anti-fungal innate immunity of human corneal epithelial cells

[2] Cynthia Brisac, et al. J Virol. Calcium flux between the endoplasmic reticulum and mitochondrion contributes to poliovirus-induced apoptosis

[3] Xiaoyu D, et al. Plasma Science and Technology. Measurement of cytoplasmic Ca2+ concentration in Saccharomyces cerevisiae induced by air cold plasma

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Abmole Inhibitor Catalog 2017




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