By David G. Lambert
David Lambert and a panel of major gurus current quite a lot of experimental protocols for learning Ca2+ signaling. those optimized thoughts disguise the extra universal purposes, together with 45Ca2+ flux measurements, and uncomplicated fluorometric expertise, in addition to extra refined equipment, together with confocal microscopy and subcellular Ca2+ imaging. There also are tools - mostly in response to fluorescence size - to figure out Ca2+ channel task and the discharge of Ca2+ from intracellular shops. Calcium Signaling Protocols deals state-of-the-art researchers effortlessly reproducible laboratory tools that give the chance to envision the calcium signaling procedure intimately in a variety of cells of animal and plant starting place.
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Extra info for Calcium signaling protocols (Methods in Molecular Biology Vol.114)
The serum serves as a block for nonspecific antibody binding. Ensure that no serum of the host of the primary antibody is used. Prior to use, clarify all diluted antibody solutions by centrifuging at 15,000g for 10 min. Proprietary mountants: Mounting media such as Fluoromount (BDH, UK), Vector Shield (Vector Labs, Peterborough, UK), and Citifluor (Agar Scientific, Essex, UK) are all useful. Antiphotobleaching properties of additives such as n-propyl gallate and paraphenylene diamine significantly prolong the lifetime of observation of many common fluorophores under the conditions of laser light excitation used.
5. As can be seen in Fig. 1, the response to 10 µM carbachol was biphasic (Fig. 1A). Also shown for comparison is a typical 340/380 nm recording (Fig. 1B) and the derived 340/380 ratio (Fig. 1C). Fura-2 Measurements in Cell Suspensions 35 Fig. 1. Carbachol increases [Ca2+]i in suspensions of SH-SY5Y cells. (A) Emission at 340 and 380 nm excitation. Note the antiparallel movement of both traces. (B) Derived 340/380 ratio and (C) [Ca 2+]i after calibration. 39, respectively. 18 arbitrary units Շ2% of cell signal.
Introduction An elevation in intracellular calcium concentration ([Ca2+]i) acts to trigger a range of cellular events including neurotransmitter release, muscle contraction, and oocyte fertilization (1,2). The pattern of elevation in [Ca2+]i and response to that elevation is dependent on the agonist and the cell type. The introduction of the calcium-sensitive dye fura-2 (3) revolutionized the measurement of [Ca2+]i in whole cell suspensions, populations of adherent cells, single cells, and in subcellular regions (see ref.
Calcium signaling protocols (Methods in Molecular Biology Vol.114) by David G. Lambert