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Fluo-8 Calcium Assay Kit - Medium Removal (ab112128)

Submit a review Q&A (8)References (1)
Functional Studies - Fluo-8 Medium Removal Calcium Assay kit (ab112128)

    Key features and details

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

    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: Mammals, Other species

    • 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.


      Fluo-8 products available


      Fluo-8 Calcium Flux Assay Kits are available in No Wash and Medium Removal variants:
      - No wash kit Fluo-8 Calcium Flux Assay Kit ab112129 is generally preferred as the workflow is simpler. It is also recommended for cell lines that are only weakly adherent and risk losing adherence when washing.

      - Medium removal kit Fluo-8 Calcium Flux Assay Kit ab112128 is recommended for the testing of compounds, receptors and calcium channels that are sensitive to the presence of cell culture media or serum. Alternatively, for cells that can tolerate it, consider using a low serum % in culture media and use No Wash Fluo-8 kit ab112129.


      Fluo-8 is also available as a standalone biochemical ab142773.

    • 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 - Fluo-8 Medium Removal Calcium Assay kit (ab112128)
      Functional Studies - Fluo-8 Medium Removal Calcium Assay kit (ab112128)
      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.

    References (1)

    Publishing research using ab112128? Please let us know so that we can cite the reference in this datasheet.

    ab112128 has been referenced in 1 publication.

    • Khandelwal N  et al. Fatty acid chain length drives lysophosphatidylserine-dependent immunological outputs. Cell Chem Biol 28:1169-1179.e6 (2021). PubMed: 33571455

    Customer reviews and Q&As

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    1-8 of 8 Abreviews or Q&A

    Question

    Questions shown with answers below

    Read More
    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

    Question

    Thanking you for answering my previous queries, hoping you would help me with this one too.
    I need to measure the intracellular calcium concentration. What equation should be used for the same.
    As ratiometric method cannot be used with Fluo 8 alone so will the following equation ie applied to single wavelength
    measurements be applicable here:

    [Ca 2+ ] = Kd * (F- Fmin ) / (F max- F)

    where Fmax and F min can be found out by using Ionomycin and EGTA respectively.
    Also if Kd of the given dye can be used as 398 or not.

    Read More
    Answer

    The lab confirmed that the formula is correct.

    I hope this is of help.

    Read More

    Question

    Hi!

    I'm interested in the product: ab112115. My intention is to use it to measure a gradient of Ca+2 by fluorescent microscopy and study cell chimotaxis at the same time. I would like to know if this product can work in this conditions: physiological conditions of pH and Tº, and if it might be toxic for the cells.
    Last but not least, I would like to know what proportion of probe is recommended to use in front of a specific concentration of calcium.

    I will deeply appreciate your help.

    Read More
    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.

    Read More

    Question

    How many plates can this kit assay? Five?
    Can the Greiner Bio-one plate, and the catalogue number is 655090 as suggested by you be used for PBMC's. My work is on calcium signalling and I am targeting the PBMC population.

    Read More
    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.

    Read More

    Question

    Would 96 well plates of corningproduct no: 3603 suit the purpose.
    Thanking you for technical support.

    Read More
    Answer

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

    Read More

    Question

    The datasheet states that black walled and clear bottom plate has to be used with the abcam product ab112128. Will the plate be provided with the kit ?

    Read More
    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.

    Read More

    Question

    The datasheet states that black walled and clear bottom plate has to be used with the abcam product ab112128. Will the plate be provided with the kit ?

    Read More
    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?

    Read More

    Question

    Dear Technical Team,




    Hope this e-mail finds you well!




    Kindly arrange to send us the Publication/ Scientific References of Fluo-8 Medium Removal Calcium Assay kit (ab112128) as desired by the customer.



    Looking forward towards your valued response on it.



    With Best Regards,

    Read More
    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.

    Read More

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