Simultaneous Monitoring Cytoplasmic Calcium Ion and Cell Surface Phosphatidylserine in the Necrotic Touch Neurons of

Calcium ions trigger many cellular events, including the release of neurotransmitters at the synaptic terminal and excitotoxic cell death. Recently, we have discovered that a transient increase in the level of cytoplasmic Ca triggers the exposure of phosphatidylserine (PS) on the surfaces of necrotic cells in the nematode . PS serves as an "eat me" signal that attracts engulfing cells to engulf and degrade necrotic cells. During the above study, we developed a microscopic imaging protocol for real-time monitoring the levels of cytoplasmic Ca and cell surface PS in touch neurons. Previously, Ca dynamics was monitored in neurons in larvae in time periods ranging from milliseconds to seconds. Methods for monitoring Ca dynamics for a relatively long period of time during embryonic development were not available, let alone for simultaneous monitoring Ca and PS dynamics. The protocol reported here utilizes a deconvolution imaging system with an optimized experimental setting that reduces photo-damage and allows the proper development of embryos during the real-time imaging process. This protocol enables the simultaneous measurement of cytosolic Ca and cell surface PS levels in necrotic touch neurons during embryonic development in a period longer than six hours. Our method provides an easy and sensitive approach to perform long-time Ca and PS recording in living animals, simultaneously or individually. This protocol can be applied to study various cellular and developmental events that involve the dynamic regulation of Ca and/or PS.