Abstract
This protocol describes the measurement of Ca(2+) sparks in intact myocytes by using a Ca(2+)-sensitive dye and imaging using laser scanning confocal microscopy. It takes advantage of spontaneous Ca(2+)-release events-sparks-using them as a measure of the activity of ryanodine receptors (RyRs). Two methodologies are described: One requires that cardiomyocytes be stimulated, preferably under voltage clamp by depolarizing pulses, until steady-state is reached, and then stimulation is stopped and Ca(2+) sparks are recorded. The second requires that cells be permeabilized and bathed in a solution to load the cell with Ca(2+) sufficient to elicit Ca(2+) sparks, but not Ca(2+) waves. These are then analyzed offline to quantify spark frequency and morphology. The advantages and disadvantages of each approach are discussed.
Original language | English |
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Pages (from-to) | 490-497 |
Number of pages | 8 |
Journal | Cold Spring Harbor protocols |
Volume | 2015 |
Issue number | 5 |
DOIs | |
Publication status | Published - 1 May 2015 |
Keywords
- Calcium/analysis
- Cytological Techniques/methods
- Fluorescent Dyes/metabolism
- Microscopy, Confocal/methods
- Myocytes, Cardiac/chemistry
- Patch-Clamp Techniques/methods
- Staining and Labeling/methods
ASJC Scopus subject areas
- General Biochemistry,Genetics and Molecular Biology