G Protein Coupled Receptors (GPCR) and ion channels are important targets in drug development. Both target classes represent membrane proteins which require convenient and robust cell-based assays for screening campaigns. Usually used as readout of such screenings, intracellular Calcium concentration implies the use of Ca2+ indicators able to meet demanding expectations of researchers engaged in assay development and screenings.
Measurement of intracellular Calcium based on fluorescent dyes
Commonly used tools to directly measure Calcium are Ca2+ chelators – in this case fluorescent dyes which directly bind Ca2+. Upon binding, the fluoresence intensity is significantly increased and can be easily measured by fluorimetric instruments. Classical dyes are Fura-2, Indo-1, Fluo-3, Fluo-4.
Fluo-8 (chemical structure, see Fig 1) can be considered as the state-of the-art indicator for measuring intracellular Ca2+ concentration, as it has 3 main benefits over classical fluorescent dyes:
- Twice as bright as Fluo-4 and four time brighter than Fluo-3
- Loading of the cell can be conducted at room temperature, meaning under less harsh conditions than with Fluo-3 and Fluo-4 which require 37°C for optimal loading.
- As especially HTS screening requires homogeneous assays (assays not requiring any wash steps to be HTS compatible), a Fluo-8 no wash kit is available as well.
Fluo-8 dye is twice brigther than Fluo-4.
Interested in testing our Fluo-8?
Just contact me with the form below.
14 responses
Please may I try the fluo-8.
Please can you also send catalogue and price details
Dear Emily,
Thank you for your message.
It is possible to test this unique Fluo-8 dye.
We are contacting you via email to give you all the information needed to perform your validation.
Regards,
Philippe
Dear bio-reactive team,
I would like to test Fluo-8 no wash assay. This sounds really interesting. Is it only possible to test the Fluo-8 dye or can I also test the no wash application?
Thank you in advance!
Dear Jessica,
Thank you for being a regular reader of our blog!
Christian will contact you very soon to further discuss with you about Fluo-8 applications.
Regards,
Philippe
Hello,
We recently acquired the Fluo8 kit and will sstart to use it very soon.,Can you please advice the best way to measure Ca2+ activity experiments? We are using mesenchymal stem cells in culture performing time lapse (acquistion every 10s).
Best,
Isabel.
Dear Isabel,
Paola will contact you directly today to assist you regarding the use of the Fluo-8 Ca2+ probe on your cellular model.
Regards,
Philippe
Dear Philippe,
I would like to test Fluo-8 both on fibroblast and on neurons. I’m very new to the field of calcium transients and I would really appreciate your help. I was wondering wether you use a free calcium medium while you add ionomycin to induce calcium release from ER and its entry from extracellular environment. Which types and concentration of drugs do you recommend? If you have an already tested protocol and you could share it t I would be very grateful.
Thanks a lot for your help and time.
Greta
Dear Dr Greta,
Thnak you for your request. I would like to confirm you that your question has been sent to the technical service of our supplier. I will come abck to ou as soon I will recevie a feedback from them.
Have a good day,
Best regards,
Frédéric
Thank you very much!
Dear Greta，
I am going to do the similar experiment as you, and I’m very new to the field, could you share your protocol with me? Thanks a lot for your help!
I used Fluo-8 in time lapse. Now I have a movie. How can I mesure fluorescence? Cell by cell or totale area? Every 4 sec and as mean grean value or intensity?
Thank you very much
Dear Dr Colitti,
Thnak you for your request on Fluo-8. I already have forwarded your technical request to techncial specialist and I will come back to you as soon as I will receive a feedback.
Have a good day,
Best regards,
Frédéric
tahnk you very much
m
Dear Philippe,
I also would like to test Fluo-8 both on fibroblast and on neurons. I’m very new to the field of calcium transients and I would really appreciate your help. I was wondering wether you use a free calcium medium while you add ionomycin to induce calcium release from ER and its entry from extracellular environment. Which types and concentration of drugs do you recommend? Dose EGTA should be in buffer？If you have an already tested protocol and I hope
you could share it with me.
Thanks a lot for your help and time.