Hi! I have no specific experiences with the calcium dye you are using. Since you have not yet received a response to your question I would like share my experiences: Probenecid is almost generally recommended to optimize dye retention in Ca imaging. To be honest, I never saw massive differences in dye retention over several hours when omitting probenecid (in neurons, neuronal cell lines and lymphocytes, imaged with FURA PE3/AM or BAPTA-Oregon Green, all at 37°). Usually Iused a very gentle washing step before transfering the cells to the microscope or spectrofluorimeter. Agressive washing can induce calcium elevations.

Good luck!

Thanks for sharing your experience! How exactely do you wash your cells, especially the lymphocytes?

Hi Christina,

I just join this group, see your question now. The key features of the colorless Ca indicator Cal-520, AM is significantly higher signal to noise ratio and much better intracellular retention (even CHO cells can be dye loaded without or much less probenecid) compare to any other existing fluorescent calcium assays. You can basically add the dye into your cell medium, incubate at 37C, 5% CO2 incubator for 2 hours (longer incubation time give better signal, and with cell medium + 5% FBS make cells healthier ), and then simply remove the dye medium, and replace with Hanks and 20 mM Hepes buffer (wash is not necessary, but you can wash once if you like) before experiment. Hope it helps. Thanks!

Jennifer

Hi Christina,

Since there is no information on molecular weight in Datasheet of Cal 520 , I would like to know how did you prepare 5mM stock? 

-Arpita

Hi Arpita,

The MW for Cal-520 AM is 1103. see the detail protocol from the following link. Thanks!

http://aatbio.com/protocol/A3300d4.pdf

Jennifer

Hi, I am planing to use Cal-520 which is why I ended reading this post. Before I start with the experiments I would like to know if there is a specific way to prepare the stock solution to ensure the dye is dissolved (vortex, sonication ...)?

Hi Alejandra,

Simply do a few times up and down pipetting is perfectly fine. Cal-520 AM is very easy to be dissolved in DMSO. You can add 200-400 uL DMSO into the 1 mg vial or 10-20 uL of DMSO into 50 ug vial to make ~2.5-5 mM stock solution. see the details below. Thanks!

https://www.aatbio.com/products/protocol/A3300d4.pdf

Thanks Jennifer!!

You are more than welcome! Alejandra

Hi all How do I calibrate the IonOptix system with the Cal-520 probe?

Thank you

Hi Morteza,

It is best to ask ionoptix's tech support. In the following link, they mentioned Fluo-4, Cal-520 should have a similar calibrate method as Fluo-4. Thanks!

https://www.ionoptix.com/products/systems/intact-muscle-chamber/

Best regards,

Jennifer