Coelenterazine h

This protocol only provides a guide, please modify it to meet your specific needs.
1. Solution Preparation
(1) Stock Solution: Dissolve Coelenterazine h in methanol or ethanol to prepare a 10mM stock solution.
Note: Unused stock solution should be aliquoted and stored at -20°C or -80°C in the dark, avoiding repeated freeze-thaw cycles.
(2) Working Solution: Dilute the stock solution with PBS or cell culture medium to 10µM.
Note: Please adjust the working solution concentration according to actual conditions, and prepare it fresh before use.
2. Cell Staining using Coelenterazine h^[1]
(1) Cell Preparation: Hela cells were transfected with a plasmid encoding the RLuc8 luciferase reporter gene using the Lipo2000 Transfection Reagent (GK20005).
(2) Add the prepared 10µM Coelenterazine h working solution to the cell culture plate. Place the plate in a 37°C, 5% CO₂ incubator and incubate in the dark for 1-4h (the specific incubation time needs to be optimized according to experimental conditions).
(3) After staining is complete, immediately detect using a fluorescence microscope. It is recommended to set the excitation/emission wavelengths to 437/466nm.
Additional Notes:
1. Coelenterazine h can be used as a luminescent substrate for the RLuc8 reporter gene in the concentration range of 1-10µM.
2. In the measurement of BRAC protein Ca²⁺ binding kinetics in solution, 5nM BRAC protein was rapidly mixed with 20µM Coelenterazine h in different concentrations of Ca²⁺ buffer, and the emission intensity change of its Venus channel (530nm) was monitored at a frequency of 1kHz. This condition can be used as a reference for cell staining experiments.

References:
[1]Saito K, Hatsugai N, Horikawa K, et al. Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level[J]. PLoS One, 2010, 5(4): e9935.