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Rhod-4 Calcium Assay Kit - No Wash (ab112157)

Submit a review Q&A (3)References (4)
Functional Studies - Rhod-4 No Wash Calcium Assay kit (ab112157)

    Key features and details

    • Assay type: Cell-based
    • Detection method: Fluorescent
    • Platform: Microplate reader
    • Assay time: 1 hr
    • Sample type: Adherent cells, Suspension cells

    Overview

    • Product name

      Rhod-4 Calcium Assay Kit - No Wash
      See all Calcium kits

    • Detection method

      Fluorescent

    • Sample type

      Adherent cells, Suspension cells

    • Assay type

      Cell-based

    • Assay time

      1h 00m

    • Species reactivity

      Reacts with: Mammals, Other species

    • Product overview

      Abcam Rhod-4 No Wash Calcium Assay Kit (ab112157) is a fluorescence-based assay for detecting intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Rhod-4 which can cross the cell membrane. Once inside the cell, the lipophilic blocking groups of Rhod-4 are cleaved by an esterase, resulting in a negatively charged fluorescent dye that stays inside the cell. Its fluorescence is greatly enhanced upon binding to calcium. When cells are stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increases the fluorescence of Rhod-4. The characteristics of its long wavelength, high sensitivity, and >250 times fluorescence enhancement make Rhod-4 the brightest red calcium indicator available in the marker, and it is an ideal tool for the measurement of cellular calcium through HTS screening. Compared to Fluo-8, Rhod-4 is more photostable, making its fluorescence imaging more robust.


      ab112157 provides an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels using HTS instrumentation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.


      Visit our FAQs page for tips and troubleshooting.

    • Notes

      This product is intended to be used for monitoring calcium fluctuations in vivo in live cells using the following HTS imaging plate readers: FLIPR™, FDSS, BMG NOVOstar™, FLexStation, ViewLux, IN Cell Analyzer or Arrayscan.

      If you would like to quantify calcium concentration in vitro using cell extracts, we recommend using Calcium Detection Kit (Colorimetric) (ab102505) or Calcium Quantification Assay Kit (ab112115), as they provide stock standards for comparison.

    • Platform

      Microplate reader

    Properties

    Images

    • Functional Studies - Rhod-4 No Wash Calcium Assay kit (ab112157)
      Functional Studies - Rhod-4 No Wash Calcium Assay kit (ab112157)
      Carbachol Dose Response was measured in HEK293 cells with ab112157 and Rhod-2. HEK293 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using ab112157, or 100 µL of Rhod-2 solution (5 µM) for 1 hour at room temperature. Carbachol (25 µL/well) was added to achieve the final indicated concentrations. The EC50 of Rhod-4 is about 0.6 µM, and that for Rhod-2 is about 0.7 µM.

    References (4)

    Publishing research using ab112157? Please let us know so that we can cite the reference in this datasheet.

    ab112157 has been referenced in 4 publications.

    • Wang Y  et al. Reduced Oligodendrocyte Precursor Cell Impairs Astrocytic Development in Early Life Stress. Adv Sci (Weinh) 8:e2101181 (2021). PubMed: 34155833
    • Jeon BH  et al. Dexamethasone Treatment Increases the Intracellular Calcium Level Through TRPV6 in A549 Cells. Int J Mol Sci 21:N/A (2020). PubMed: 32033337
    • Schick R  et al. Electrophysiologic Characterization of Developing Human Embryonic Stem Cell-Derived Photoreceptor Precursors. Invest Ophthalmol Vis Sci 61:44 (2020). PubMed: 32991686
    • Pandey M  et al. A central role for R7bp in the regulation of itch sensation. Pain 158:931-944 (2017). PubMed: 28134655

    Customer reviews and Q&As

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    1-3 of 3 Abreviews or Q&A

    Question

    Questions shown with answers below

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    Answer

    Thank you for your inquiry.

    1. if the kits for measuring intracellular calcium mobilization can work with mouse cell lines, specifically MLE-12 cell line?--Yes, as long as the cells contains calcium channels and Gq receptors

    2. Also, how does one decide if we should use the Rhod-4 or Fluo-8 kits, and whether or not to remove the cell growth medium?-- Fluo-8 kit is more sensitive compare to Rhod-4 kits. If your cells don't have GFP, use fluo-8 kits. Rhod-4 kits is mainly for cell lines have GFP proteins, so you can use red fluorescence to overcome the green fluorescence from GFP. If your compounds are sensitive to serum such as growth factors, you will have to remove medium.

    3. I would also like to make sure, are the kit components the same for the no wash and medium removal no wash kits?--No, the amount of dye and component B are different.

    4. And each kit can assay one 96-well plate?--Yes.

    I would recommend ab112129 first. Also, the calcium response is a transient response, so you do need a fluorescent plate reader with bottom read mode and inside dispenser such as Flexstation or fluorescent microscope to perform calcium experiments.

    Since this product has not yet been tested in mouse, we have our AbTrial program which allows researchers to test our products in untested applications or species without financial risk. Details regarding the abTrial program are given below:
    https://www.abcam.com/index.html?pageconfig=resource&rid=11998

    Please let me know if you are interested in testing one of these calcium assay kits in mouse or if you have any other questions. I look forward to your reply.

    Would you use an Abcam personal shopper to help you find the right products for your research needs? Answer the 1 question “yes or no” survey below. Thank you for your input!
    https://www.abcam.com/shopper

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    Question

    Dear xxxxx,

    thanks for your email and your support! So I take it that the presence of zinc would also contribute to the signal and ENHANCE the signal (and not reduce it, right?)?

    I had a look at the assay you’re mentioning… it seems it needs cells to work (some kind of activation process via cell esterases)????? This is clearly not what we’re looking for, since we are not working ith cells (simply Ca in solution).

    Would be great if you could confirm this.

    Kind regards,

    Read More
    Answer

    Thank you for getting back to me.

    Yes, the signal would be expected to be enhanced in the presence of zinc.

    And in reply to your following question, yes the Fluo-8 (ab112129) and Rhod-4 (ab112157) would not be suitable for your samples. I am sorry for the confusion caused.

    If you have any further questions, please do not hesitate to ask.

    Read More

    Question

    Dear Technical support team of abcam,

    Recently I ordered 'Rhod-4 No Wash Calcium Assay Kit (ab112157)' from abcam for GPCR calcium flux assay.

    In the protocol document HHBS (1x Hank's with 20mM Hepes Buffer, pH 7.0) is indicated as washing buffer as well as assay compound solutionin order to minimize background fluorescence.

    My question is that can HHBS be replaced with DPBS?

    Does DPBS cause any other problems in the use of your product for GPCR calcium flux assay?


    I appreciate in advance your kind reply.

    Thank you.


    ---------------------

    Read More
    Answer

    Thank you for your inquiry.

    I can confirm that you can use PBS instead of HHBS.

    It is essential that20mM Hepes Buffer is added to the PBS (as it is added to the HHBS).

    I wish you good luck with your experiments.

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