Journal
STAR PROTOCOLS
Volume 2, Issue 3, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.xpro.2021.100618
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Funding
- NIH/NHLBI [NIH/DE019245]
- [R35-HL150778]
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This protocol details the steps to visualize and detect Ca2+ puffs in HEK-293 cells using TIRF microscopy, offering high-speed imaging with improved signal-to-background ratio. Common pitfalls encountered while recording Ca2+ puffs using TIRF microscopy are also highlighted.
This protocol outlines steps to visualize and detect Ca2+ puffs following photo -liberation of caged inositol-1,4,5-trisphosphate (IP3) from HEK-293 cells express-ing only the native IP3R type 1 receptor using total internal reflection fluores-cence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with a higher signal-to-background ratio. Additionally, we shed light on commonly encountered pitfalls, which should be considered while recording Ca2+ puffs using TIRF microscopy.For complete details on the use and execution of this protocol, please refer to Emrich et al. (2021) and Lock et al. (2015a).
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