Fluo-8 Calcium Flux Assay Kit - No Wash (ab112129)

Thank you for your enquiry by telephone yesterday and for your patience while I contacted the laboratories.

They have confirmed that this is correct, the Pluronic F127 Plus component is red in color in the tube.

The fluorescence that is read for this assay is green.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

Thank you for your message and I am sorry to hear you have had difficulty obtaining results from ab112129 Fluo-8 No Wash Calcium Assay kit

The only fluorescence microplate readers recommended for ab112129 Fluo-8 No Wash Calcium Assay kit are the ones listed in the protocol booklet. The kit has been tested using the Novostar.

Clariostar has everything except the built in pipetter which is essential for measuring calcium response accurately due to the transient property of the calcium signal. Depending on dose of the agonist, the peak response is few seconds to 20 seconds, so one will need to add the compound by instrument and read the fluorescent signal simultaneously every second for ˜1-2 minutes. see the example curve using Novostar on the kit datasheet.

If you need to use the Clariostar, we can recommend to try adding the compound (such as control and treatment) one column at a time, and read as soon as possible for a 30 seconds kinetics, and use the highest point see if you obtain anything. However, optimal results would be obtained from the platereaders listed in the protocol booklet which include the in built pipetter (FLIPR™, FDSS, BMG NOVOstar™, FlexStation, ViewLux, IN Cell Analyzer or Arrayscan).

I hope this will be helpful. Should you use one of the recommend platereaders and still experience difficulties, please let me know as the kit will be covered by the Abpromise guarantee. UPDATE from manufacturer of CLARIOstar: The CLARIOstar can do FLUO-8 calcium flux, but it can’t screen a plate of agonists like the NOVOstar, only specialized instruments like are mentioned above can do that. The CLARIOstar can do one injection event at the point of measurement. Therefore, it can be used for calcium flux dose response curves by increasing the injection volumes of one particular agonist of interest; or it can be used to look for antagonists, inhibitors, or blockers, which are plated first with the cells and then stimulated with one particular agonist (i.e. epinephrine). The CLARIOstar does require a specific injection ‘spoon’ for simultaneous injection and measurement, which is needed for calcium flux assays. Please contact manufacturer for assistance.

Use black wall clear bottom 96 well cell culture treated plates.

Yes, the kit contains Probenecid. No additional Probenecid is needed for the kit.

The Ca response is a transient response (a couple of the seconds to 10-20 seconds), it is almost impossible to pipette the compounds into the plate, and read the response. Yes, you do use the multichannel pipette to add the dye into the plate, and let incubate for 1 hour at 37C or room temperature. And then add the agonist using the fluid injector within the instrument. and read the fluorescence every second for 90 seconds. You can try a single column a time, and read for 30 second (1 sec a point), see if you can get some response at high dose. Or simply try under microscope, take a picture before the compound addition, and then add compound, take a few pictures see how it goes. Hope it helps.

Yes, the kit can be used for all the mammalian cells - the lab did confirm this.

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.

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Thank you for contacting us.


The kit uses our water soluble and heat stable probeneic to prevent dyes from leaking out of the cells.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you very much for your email.

I realize that there was a typos in my last email. The kD value I should have written is 320nm. However I also contacted laboratory after receiving your reply - they have informed me that the kD value is 389nm.

I am very sorry about the confusion. Please record 389 as kD value for this product. If this discrepancy in kD value has affected your results, please let me know we can replace the product we have sent.

I hope this information will be helpful. Should you have more question please do not hesitate to contact me.

Thank you for contacting us.

It seems that you have asked similar question last year in October and we provided the 230nm as dissociation constant. The 230 nm Kd value is available to us from our collaborator who developed this product for us. Do you think this value does not seem realistic? I can ask the laboratory again if there is any doubt.

I will look forward to hearing from you soon. Please do not hesitate to contact us if you need any more advice or information.