Developmental Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1380 Views May 20, 2024

Calcium signalling in the endocardium is critical for heart valve development. Calcium ion pulses in the endocardium are generated in response to mechanical forces due to blood flow and can be visualised in the beating zebrafish heart using a genetically encoded calcium indicator such as GCaMP7a. Analysing these pulses is challenging because of the rapid movement of the heart during heartbeat. This protocol outlines an imaging analysis method used to phase-match the cardiac cycle in single z-slice movies of the beating heart, allowing easy measurement of the calcium signal.

0 Q&A 2370 Views Dec 5, 2021

Visualizing the function of pancreatic β-cells in vivo has been a long-sought goal for β-cell researchers. Unlike imaging of β-cells in mammalian species with conventional positron emission tomography and single-photon emission computed tomography, which only provides limited spatial-temporal resolution, transparent zebrafish embryos are a unique model that allows high-resolution fluorescent imaging of β-cells in their native physiological microenvironment in vivo. Here, we detail a protocol for real-time visualization of individual β-cell function in vivo in a non-invasive manner, through combination of a novel transgenic zebrafish reporter line Tg (ins:Rcamp1.07) with both a commercial spinning-disc confocal microscope and an in-house developed super-resolution microscope (2P3A-DSLM). The protocol described here allows for the longitudinal monitoring of dynamic calcium activities from heterogeneous β-cells in early developing zebrafish embryos and is readily adaptable for use in imaging other important processes in islet biology, as well as screening new compounds that can promote β-cell function or maturation using a living whole organism system.

0 Q&A 4998 Views May 20, 2019
Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous monitoring can offer additional insights into P2 receptor physiology. This protocol takes advantage of the overlapping fluorescence spectra between the ATP-detection substrate luciferin and calcium indicator dye Fura2. Mammalian cells are loaded with Fura2-AM and live-cell recordings are acquired in the presence of a luciferin-luciferase imaging solution. This protocol allows to study stimulus-induced ATP release and directly relate changes in extracellular ATP concentration to observed calcium responses.

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