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(Synonyms: Fluo-4 Acetoxymethyl ester) 目录号 : GC30231

A fluorescent calcium indicator

Fluo-4 AM Chemical Structure

Cas No.:273221-67-3

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50ug
¥900.00
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100ug
¥1,350.00
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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

Cell experiment:

For measuring fluorescence from cells in suspension, dilutions to 2 to 3×106 cells are made, from cultures of rat basophilic leukemia (RBL) cells. Cells are incubated in suspension in 1 mM dye (including Fluo-4 AM) for 30 min at 37°C. Cell suspensions are then transferred to cuvets for measurements of fluorescence emission intensity by spectrofluorometer[1].

References:

[1]. Gee KR, et al. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium. 2000 Feb;27(2):97-106.

产品描述

Fluo-4 acetoxymethyl ester (Fluo-4 AM) is a cell-permeable fluorescent calcium indicator.1 It is cleaved by intracellular esterases to release Fluo-4, which binds to calcium with a Kd value of 345 nM and displays excitation/emission maxima of 490/520 nm, respectively. Fluo-4 AM has been used to measure intracellular calcium levels in live cells by fluorescent microscopy or flow cytometry.1,2

1.Gee, K.R., Brown, K.A., Chen, W.N., et al.Chemical and physiological characterization of fluo-4 Ca2+ - indicator dyesCell Calcium27(2)97-106(2000) 2.Vines, A., McBean, G.J., and Blanco-Fernández, A.A flow-cytometric method for continuous measurement of intracellular Ca2+ concentrationCytometry A.77(11)1091-1097(2010)

Chemical Properties

Cas No. 273221-67-3 SDF
别名 Fluo-4 Acetoxymethyl ester
Canonical SMILES O=C(OCOC(C)=O)CN(C1=CC=C(C2=C3C=C(F)C(C=C3OC4=C2C=C(F)C(OCOC(C)=O)=C4)=O)C=C1OCCOC5=CC(C)=CC=C5N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O
分子式 C51H50F2N2O23 分子量 1096.94
溶解度 Methanol: soluble 储存条件 Store at -20°C, protect from light
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溶解性数据

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1 mg 5 mg 10 mg
1 mM 0.9116 mL 4.5581 mL 9.1163 mL
5 mM 0.1823 mL 0.9116 mL 1.8233 mL
10 mM 0.0912 mL 0.4558 mL 0.9116 mL
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Research Update

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp

Cytosolic Ca2+ plays a key role in plant development. Calcium imaging is the most versatile method to detect dynamic changes in Ca2+ in the cytoplasm. In this study, we obtained viable protoplasts of pulp cells by enzymatic hydrolysis. Isolated protoplasts were incubated with the small-molecule fluorescent reagent (Fluo-4/AM) for 30 min at 37 °C. The fluorescent probes successfully stained cytosolic Ca2+ but did not accumulate in vacuoles. La3+, a Ca2+ channel blocker, decreased cytoplasmic fluorescence intensity. These results suggest that Fluo-4/AM can be used to detect changes in cytosolic Ca2+ in the fruit flesh. In summary, we present a method to effectively isolate protoplasts from flesh cells of the fruit and detect Ca2+ by loading a small-molecule calcium fluorescent reagent in the cytoplasm of pulp cells.

Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis

Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators.
Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel.
Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2.
Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Monitoring intracellular calcium ion dynamics in hair cell populations with Fluo-4 AM

We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca(2+) signals in populations of hair cells. The bundle Ca(2+) signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca(2+) entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca(2+) chelators or blocking Ca(2+) entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca(2+) decrease, which could serve to stimulate the hair cell's tip-link regeneration process. Measurement of bundle Ca(2+) with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca(2+) signaling in the hair cell.

Reliable measurement of free Ca2+ concentrations in the ER lumen using Mag-Fluo-4

Synthetic Ca2+ indicators are widely used to report changes in free [Ca2+], usually in the cytosol but also within organelles. Mag-Fluo-4, loaded into the endoplasmic reticulum (ER) by incubating cells with Mag-Fluo-4 AM, has been used to measure changes in free [Ca2+] within the ER, where the free [Ca2+] is estimated to be between 100 μM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [Ca2+] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca2+ measured in vitro (KDCa ?22 μM). Using an antibody to quench the fluorescence of indicator that leaked from the ER, we established that the affinity of Mag-Fluo-4 within the ER is much lower (KDCa ?1 mM) than that measured in vitro. We show that partially de-esterified Mag-Fluo-4 has reduced affinity for Ca2+, suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for Ca2+ that are both appropriate for the ER lumen and capable of reporting a wide range of free [Ca2+].