Fluorescent Dyes for Calcium Imaging Experiments
Calcium ions (Ca2+) play important mobile physiology roles in sign transduction pathways, in neurotransmitter launch, in contraction of all muscle cell sorts, as enzyme cofactors, and in fertilization. Extracellular calcium can be essential for sustaining the potential distinction throughout excitable cell membranes, as is nicely often called a requirement for correct bone formation. Analyzing calcium flux utilizing reside cell calcium imaging methods is essential to understanding mobile operate and dysfunctions that could be elements of illness.
Chemical calcium indicators and sensors are small molecules that bind calcium ions by way of chelation. Many are primarily based on widely-used calcium chelators like EGTA, (ethylene glycol-bis(β-aminoethyl ether), or on chelators with enhanced calcium ion (Ca2+)specificity or pH stability, reminiscent of BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). These dyes are sometimes modified with acetoxymethyl esters (AM), with the intention to render the molecule lipophilic and to permit straightforward entrance into the cell. As soon as contained in the cell, mobile esterases will free the carboxyl teams,enabling Ca2+ by the indicator. Binding of a Ca2+ ion to a fluorescent indicator molecule leads both to a rise in quantum yield of fluorescence or an emission/excitation wavelength shift.
Conventional and Novel Calcium Imaging Probes
Fura-2: A UV light-excitable, ratiometric Ca2+ indicator that could be a frequent dye for ratio-imaging microscopy. Upon binding Ca2+, Fura-2 reveals an absorption shift that may be noticed by scanning the excitation spectrum between 300 and 400 nm, whereas monitoring the emission at ~510 nm.
Indo-1: A UV light-excitable Ca2+ indicator that’s the popular calcium sensor for movement cytometry that makes use of a single laser for excitation. The emission most of Indo-1 shifts from ~475 nm in Ca2+-free medium to ~400 nm when the dye is saturated with Ca2+.
Quin-2: Shows excessive selectivity for calcium. This calcium indicator shouldn’t be affected by sodium gradients, membrane potential vary, or intracellular pH, and shows excessive affinity for Ca2+ ions .Quin-2 is delicate sufficient to observe low ranges of calcium, reminiscent of these present in resting cells. Excitation and emission maxima are 339/492 nm, respectively.
Fluo-3: A fluorescence indicator of intracellular calcium (Ca2+) utilized in movement cytometry and confocal laser scanning microscopy. Upon binding of Ca2+, its fluorescence will increase sharply with an emission most at 525nm and is appropriate for detection utilizing a fluorescein isothiocyanate (FITC) channel.
Close to-Infrared Calcium Dyes: BioTracker NIR Ca2+ dyes (SCT021, SCT022, SCT023) are far-red fluorescent calcium indicators. These dyes adjustments fluorescent depth vastly (1000-fold) when it binds to calcium in residing cells. The benefit of those lengthy wavelength dyes is larger tissue penetration, decrease phototoxicity together with multicolor imaging utilizing Hoechst, Fluorescein, Rhodamine, GFP, YFP and RFP and many others.
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Associated Merchandise
– “calcium live”