The Fluorescence-Based Calcium Assay &the GCaMP Family of GECIs
This article discusses the Fluorescence-based calcium assay and the GCaMP family of GECIs. It also covers the PBX Signal Enhancer. The Fluorescence-based calcium assay requires the removal of media prior to detection. It is a rapid method for calcium determination and is compatible with a wide range of cell types. You can also use it in conjunction with other calcium assays to assess the concentration of various calcium ions in aqueous solutions.
GCaMP family of GECIs
GECIs are fluorescent compounds engineered to change their fluorescence based on the presence of a single fluorophore. The GCaMP family of GECIs consists of a circularly permuted form of the green fluorescent protein (GFP) linked to calmodulin (CaM) at either its N-terminus or C-terminus. The calmodulin/GFP peptides create a calcium-dependent increase in the fluorescence of the GFP.
Several GECIs have been developed to mimic the properties of Ca2+. The GCaMP family contains two members, GCaMP2 and GCaMP1.6. Both have high affinity for Ca2+ and their Kds are low (167 nM).
Fluorescence-based calcium assay
A fluorescent-based calcium assay can be used to detect the presence of calcium in cells. The fluorescent response of calcium to a calcium compound is transient and peaks in a few seconds to 20 seconds. Detection is performed by reading the fluorescent signal every second for up to two minutes. The fluorescence-based calcium assay can also be used with cell extracts. Several parameters are needed to optimize the assay.
A fluorescence-based calcium assay detects intracellular calcium mobilization in cells by tracking the release of Ca2+ into the cytoplasm. The calcium-sensitive dye used in this assay crosses the cell membrane and binds to the freed calcium. The resulting change in fluorescence is proportional to the amount of intracellular calcium. Some cells have a specific anion transporter which is particularly active. Probencid is also required to retain the calcium-sensitive dye within the cell.
PBX Signal Enhancer
For the PBX Signal Enhancer for calcium assasy, mix 10 ml of the product with 90 ml of 1X Calcium Assay buffer. Alternatively, probenecid can be used in the dye-loading procedure. While probenecid is not required, it enhances the retention of the dye in the cells. For preparing 1X Dye-loading Solution, dilute probenecid stock in 1X PBX Signal Enhancer and add to a cell plate.
The PBX Signal Enhancer solution contains probenecid, glucose, sodium chloride, and KCl. The pH of the solution is adjusted to 7.00. Calcium measurements were performed using excitation and emission filters of 470/95 nm. The PBX Signal Enhancer for calcium assay is recommended for a wide range of applications, including the study of phosphorus, magnesium, and calcium.