Overview

  • Product name
    Fluo-8 Calcium Assay Kit - Medium Removal
    See all Calcium kits
  • Detection method
    Fluorescent
  • Sample type
    Adherent cells, Suspension cells
  • Assay type
    Cell-based
  • Species reactivity
    Reacts with: Other species, Mammals
  • Product overview

    Abcam's Fluo-8 Calcium Assay Kit - Medium Removal (ab112128) is a fluorescence-based assay for detecting intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fluo-8 which can cross the cell membrane. Once inside the cell, the lipophilic blocking groups of Fluo-8 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 Fluo-8. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement make Fluo-8 the brightest green calcium indicator available in the marker, and it is an ideal tool for the measurement of cellular calcium through HTS screening.


    ab112128 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

  • Carbachol Dose Response was measured in HEK293 cells with ab112128 and a Fluo-4 assay kit. HEK293 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The growth medium was removed, and the cells were incubated with 100 µL of dye-loading solution using ab112128 or the Fluo-4 kit for 1 hour at room temperature. Carbachol (25 µL/well) was added to achieve the final indicated concentrations. The EC50 of ab112128 is about 1.2 µM.

Protocols

References

ab112128 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question
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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Answer

Thank you for contacting us and your interest in our products.

The Calcium Quantification Assay kit (Fluorometric), ab112115, is intended for use with solution based samples. For your experiment, it may be better to have a look at the Fluo-8 Medium Removal Calcium Assay kit (ab112128) or the Fluo-8 No Wash Calcium Assay kit (ab112129) to see if you think these would be suitable for you.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to contact us again.

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Answer

Thank you for contacting us.

The reagents in ab112128 are sufficient for one 96-well plate, not five.

Regarding the Greiner Bio-one plate, catalogue number is 655090, you could use it, however it is much better to use Poly-D-Lysine coated black wall/clear plate from BD (cat# is 356640) for suspension cells.

For calcium signaling, you will need the instruments such as FLIPR, Flexstation, Novostar etc which have internal dispensing system for agonist due to the calcium signal is transient effect.

Please let me know if you have any other questions.

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Answer

Yes, that plate will be suitable.
Please let me know if you have any other questions or concerns.

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Answer

Following up my previous reply, I have the following information from the developer of the assay:

The calcium assay in cells is a transient response, so you will need a bottom-read fluorescent plate reader with a built-in pipetting system such as FLIPR, Flexstation (Molecular Devices) or Novostart (BMG). Any black wall/clear bottom 96-well cell culture treated plate will be fine. We use Greiner Bio-one plate, and the catalogue number is 655090.

Please let me know if you have any other questions.

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Answer

Thank you for your inquiry.

No, the plate is not included with the kit. If you need a recommendation for a plate, could you please tell me if you are working with adherent cells or non-adherent cells?

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Answer

Thank you for your inquiry.

We do not know of any publications that used ab112128 at this point in time. As soon as we receive news about a publication we will up-date the datasheet.

I would like to reassure your customer that we guarantee our products to work as stated on the datasheet.

I hope this information is helpful and wish your customer good luck with their research.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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