When performing calcium flux assay in living cells with fluorescent calcium indicators, can you use a fluorescent plate reader that does not have a kinetic reading function or a built-in liquid handler?
Posted June 13, 2019
Yes, you can consider using Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit (Cat# 36312).
Most calcium flux assays performed require fluorescent plate readers with a built-in liquid handling system or kinetic read capability (e.g. FlexStation, FLIPR) due to the transient and fast reaction of calcium movement inside cells upon stimulation. Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit provides an alternative method which allows you to conduct calcium flux assays using a simple fluorescent microplate reader with bottom read mode.
Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit contains a fluorescent calcium indicator Fluo-8E™ AM and a proprietary formulation to enhance signals. The Fluo-8E™ has a similar spectrum to Fluo-3 and Fluo-4, Ex/Em=485 nm/525 nm. Fluo-8E™ AM can passively diffuse across intact cell membranes. Once inside the cells, the lipophilic blocking groups of Fluo-8E™ AM are cleaved by esterase, resulting in a negatively charged fluorescent dye that stays inside the cells. Upon calcium chelation, Fluo-8E™ AM’s fluorescence intensity enhances >100 times and can last for 1-2 minutes with minimal signal decrease. These characteristics make the Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit ideal for measuring cellular calcium using fluorescence microplate readers that do not have a fluid transfer or kinetic reading mode capability. The sensitivity and accuracy of the results gathered when using the Cell Meter™ Endpoint Calcium Assay Kit is comparable to the results gathered when using a microplate reader with kinetic reading capabilities.
Additionally, the Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Kit does not require the use of any organic anion transporter inhibitors, such as probenecid, or wash steps. Probenecid is an organic anion transporter inhibitor used to improve the cellular retention of calcium indicators after dye loading. Not only does preparing a probenecid solution add more steps to the assay protocol, it also shows apparent cell cytotoxicity. Furthermore, probenecid also changes the calcium flux kinetics of a cyclic nucleotide gated ion channel and decreases the channel’s response to agonists of GPCR. Using Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Kit will mitigate such negative effects commonly associated with calcium flux assays using probenecid.
Fluo-3, AM *Bulk package* *CAS 121714-22-5*