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    products/assay-kits/rhod-4-calcium-assay-kit-no-wash-ab112157.pdf

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Signal Transduction Signaling Pathway Calcium Signaling Other
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Rhod-4 Calcium Assay Kit - No Wash (ab112157)

  • Datasheet
  • SDS
  • Protocol Booklet
Submit a review Q&A (3)References (4)

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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

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    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

    • Storage instructions

      Store at -20°C. Please refer to protocols.
    • Components 1 kit
      10X Pluronic® F127 Plus 1 x 1ml
      HHBS 1 x 9ml
      Rhod-4 1 vial
    • Research areas

      • Signal Transduction
      • Signaling Pathway
      • Calcium Signaling
      • Other
      • Signal Transduction
      • Metabolism
      • Vitamins / Minerals
      • Kits/ Lysates/ Other
      • Kits
      • Cell Signaling Kits
      • Ions and Metal Assay Kits
      • Kits/ Lysates/ Other
      • Kits
      • Cell Metabolism Kits
      • Ion and metal assay kits
      • Kits/ Lysates/ Other
      • Kits
      • Cell Signaling Kits
      • Calcium assay kits
    • Relevance

      Calcium is essential for all living organisms, where Ca2+ sequestration and release into and out of the cytoplasm functions as a signal for many cellular processes. 99% of calcium is found in bones and teeth with the remaining 1% found in the blood and soft tissue. Serum calcium levels are tightly controlled (8.4-11.4 mg/dL) and any variation outside this range can have serious effects. Calcium plays a role in mediating the constriction and relaxation of blood vessels, nerve impulse transmission, muscle contraction, and hormone secretion. Calcium ion channels control the migration of calcium ions across cell membranes, permitting the activation and inhibition of a wide variety of enzymes. Causes of low calcium levels include chronic kidney failure, vitamin D deficiency, and low blood magnesium levels that can occur in severe alcoholism.
    • Alternative names

      • Ca++
      • Ca2+

    Associated products

    • Related Products

      • Ryanodine, Ca2+ release modulator (ab120083)
      • Ionomycin Ca2+ Salt, Ca2+ ionophore (ab120116)
      • Thapsigargin, Ca2+-ATPase inhibitor (ab120286)
      • BAPTA-AM, Ca2+ chelator (ab120503)

    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.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    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

    Show All Reviews Q&A
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    1-3 of 3 Abreviews or Q&A

    Question

    Questions shown with answers below

    Read More

    Abcam community

    Verified customer

    Asked on Jan 18 2013

    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

    Read More

    Abcam Scientific Support

    Answered on Jan 18 2013

    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

    Abcam community

    Verified customer

    Asked on Sep 21 2012

    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

    Abcam Scientific Support

    Answered on Sep 21 2012

    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

    Abcam community

    Verified customer

    Asked on Aug 02 2012

    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.

    Read More

    Abcam Scientific Support

    Answered on Aug 02 2012

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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