Overview

  • Product name
    Calcium Assay Kit (Luminometric)
    See all Calcium kits
  • Detection method
    Luminescent
  • Sample type
    Adherent cells, Suspension cells
  • Assay type
    Cell-based
  • Product overview

    Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). This kit uses a highly calcium-sensitive and membrane-permeable coelenterazine analog as a calcium indicator for the cells that are transfected with apoaequorin gene. Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria that has been used extensively as a calcium indicator in cells. The aequorin complex emits blue light when bound to calcium ions. The luminescence intensity is directly proportional to the Ca2+ concentration.

    ab112114 is much more sensitive than the fluorescence-based calcium assays. This kit provides an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation. It might be useful for monitoring the intracellular calcium mobilization in a specified compartment given that recombinant apoaequorin proteins can now be targeted to specific organelles, cells and tissues. ab112114 is more sensitive than the fluorescent calcium assays.

    Visit our FAQs page for tips and troubleshooting.

  • Notes

    ab112114 should be stored desiccated.

  • Platform
    Microplate reader

Properties

Images

  • ATP Dose Response on CHO-aeq cells. CHO cells stably transfected with apoaequorin were seeded overnight at 50,000 cells/100 µL/well in a white wall/clear bottom 96-well plate. The growth medium was removed and the cells were incubated with 100 µL of dye-loading solution using ab112114 for 3 hours at room temperature and protected from light. ATP (25 µL/well) was added to achieve the final indicated concentrations. The EC50 of ATP is about 0.8 µM.

Protocols

References

ab112114 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Calcium is essential for all living organisms.And because Calcium is an earth alkali metal but not a species specific protein, it should work with rat samples, too.

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Question
Answer

You are welcome. We are assuming the recovery period will be sufficient to allow demonstration of differences among treatments or sample types. You might consider comparing samples +/- the recovery period.

I just found a paper that discusses another approach, but the preparation of the culture surface appears to be fairly complicated. The study of microglia response to harvest does not appear to have any discussion of calcium flux but it looks interesting, in any case.

Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method. Biomaterials. 2001 Jun;22(11):1213-23.

http://144.206.159.178/ft/114/35315/605413.pdf

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Answer

Thank you for contacting us.

The developer of the assay had two suggestions which may be useful. The aim in each case is to allow the cells to recover from the removal from the culture plates. The second may not be acceptable if you have a specific treatment protocol or lack the instrumentation.

1. Simply harvest the cells with EDTA (not trypsin, if you are going to do the experiment with in a day) to keep the membrane receptors intact, and then add the suspension into 96-well Poly-D lysine plate, allowing the cells recover at least 6 hours before the experiment.

2. Alternatively, you can also incubate the use cells in suspension after the harvest from the 6-well plates with coelentrazine loading solution (per protocol) for 4-6 hours, and then dispense the cells into the 96-well plate that contains your compound and control. You will need an accurate dispensor within the instrument and the cell suspension must be uniform while dispensing into the wells.

Please contact me if you would like to discuss this further.

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Question
Answer

Thank you for your email. Unfortunately this kit is not suitable for tissues. The assay is aequorin based, and you will need to transfect the aequorin gene into the cells before the experiment. Aequorin binds Ca2+ and emits blue light, therefore unless cells are expressing the Aequorin there will be no fluorescence. It is also necessary to add coelenterazine into the culture medium of the cells to obtain a functional aequorin protein, as the recombinant form is an apoprotein. We have another calcium detection kit which may be more suitable, please find the link below: ab102505 Calcium Quantification Assay kit (Fluorometric) Click here (or use the following: https://www.abcam.com/index.html?datasheet=102505). The kit has been tested on lysates, the buffer provided in the kit can be used to lyse tissue samples as well. The lysis efficiency is increased by using a Dounce homogenizer in this case. The rest of the steps can be followed as in data sheet. Please do not hesitate to contact me should you have further questions.

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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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