Fluo-3 AM Probes
The membrane-permeable AM ester form of Fluo-3 can be loaded into cells by incubation. Fluo-3 AM ester itself does not bind Ca2+, but once the dye enters the cell, it is easily hydrolyzed to Fluo-3 by endogenous esterase. An important application of Fluo-3 AM ester is its use in high-throughput drug screening. Fluo-3 AM is one of the most commonly used fluorescent probes to detect intracellular calcium ion concentration. The molecular formula is C51H50Cl2N2O23 and the molecular weight is 1129.85. Compared with Fura-2, Fluo-3 has the following advantages: on the one hand, it can be excited by the 488nm excitation light of an argon ion laser (argon-ion laser) to facilitate detection; on the other hand, Fluo- 3 Fluorescence changes stronger after binding with calcium ions, that is, the detection of changes in calcium ion concentration is more sensitive; at the same time, the binding ability of Fluo-3 and calcium ions is weaker, which can detect higher concentrations in cells than Fura-2. Calcium ion level; In addition, it reacts more accurately to the instantaneous changes of calcium ions in the cell, reducing the lag in fluorescence changes caused by the slow dissociation of calcium ions. Fluo-3 AM is a fluorescent dye that can penetrate cell membranes. Fluo-3 AM's fluorescence is very weak, and its fluorescence will not increase with the increase of calcium ion concentration. After entering the cell, Fluo-3 AM can be cleaved by intracellular esterase to form Fluo-3, which is then retained in the cell. Fluo-3 can be combined with calcium ions, which can produce strong fluorescence when combined with calcium ions. The maximum excitation wavelength is 506nm and the maximum emission wavelength is 526nm. The recommended excitation wavelength is 488nm and the emission wavelength is 525-530nm.
Figure 1. Structure of Fluo-3 AM.
Fluo-3 is a fluorescent indicator of intracellular calcium (Ca2+). It is used to measure Ca2+ in living cells using visible light excitation (compatible with an argon laser source operating at 488 nm) in flow cytometry and confocal laser scanning microscopy. Fluo-3 is a basic non-fluorescent compound, but when combined with Ca2+, its fluorescence will increase sharply and its maximum emission will occur at 525 nm. It is suitable for the design of fluorescein isothiocyanate (FITC) measurement. Routine detector. This large change in fluorescence, coupled with good photon yield, provides very high contrast so that tiny Ca2+ release events inside cells called "calcium sparks" can be detected. Although the salt of fluo-3 cannot penetrate cells, it can be loaded using its acetoxymethyl (AM) ester derivative. Once in the cell, non-specific esterases cleave esters, effectively capturing fluo-3. Because calcium is a key secondary messenger within cells, the special properties of fluo-3 allow researchers to study the time-resolved kinetics of intracellular signal transduction in various cells.
Figure 2. Structure of Fluo-3 Probes.
Introductions
Fluo 3 is almost non-fluorescent if it exists as a free ligand, but when it is combined with calcium ion Ca2+, its fluorescence will increase 60 to 80 times. It is currently the most commonly used calcium ion fluorescent probe. The laser confocal fluorescence microscope has an argon laser, so Fluo 3 can be widely used in this microscope. This long wave from the fluorescent signal also facilitates reducing light damage to the sample cells. Fluo 3 can also be used to detect cleavable chelated calcium or other forms of calcium under UV light. Fluo 3-AM is an acetomethyl derivative of Fluo 3, which can easily enter cells through culture. Fluo 3-AM (Calcium Fluorescent Probe) needs to be formulated with anhydrous DMSO. Fluo 3-AM is a fluorescent dye that penetrates cell membranes. After entering the cell, Fluo 3-AM can be cleaved by intracellular esterase to form Fluo 3, which is then retained in the cell. Fluo 3 can be combined with calcium ions. Combining calcium ions can produce strong fluorescence, with a maximum excitation wavelength of 506 nm and a maximum emission wavelength of 526 nm.
References:
- Latt, SA.; et al. Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. Journal of Histochemistry and Cytochemistry. 1975, 23 (7): 493–505.
- Cheng, H.; et al. Calcium Sparks - Elementary Events Underlying Excitation-Contraction Coupling in Heart-Muscle. Science. 1993, 262 (5134): 740–744.
Applications
