Fluo 4 acetoxymethyl ester

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, France

Fluo-4 acetoxymethyl ester is a fluorescent calcium indicator compound used for detecting and measuring intracellular calcium levels. It is a cell-permeant compound that can be loaded into cells, where it undergoes hydrolysis to produce the active, calcium-sensitive fluorescent dye.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Fluo-4 (326 citations) Fluo-4 acetoxymethyl (AM) ester (17 citations) Fluo−4 acetoxymethyl ester (Fluo−4 AM) (10 citations) Fluo-4 acetoxymethyl ester (Fluo-4) (2 citations) Fluo-4 acetoxymethyl (2 citations)

33 protocols using fluo 4 acetoxymethyl ester

Quantifying Glucagon-Like Peptide-1 Receptor Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

For tissue culture Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, hygromycin-B, and Fluo-4 acetoxymethyl ester were used (Invitrogen Carlsbad, CA). AlphaScreen reagents, ¹²⁵I-Ex(9–39), ¹²⁵I-hGLP-1 and 384-well ProxiPlates were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). hGLP-1, pGLP-1, eGLP-1 and Ex-4 were purchased from GL Biochem (Shanghai) Ltd. (Shanghai, China). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or BDH Merck (Melbourne, VIC, Australia) and were of an analytical grade. The parental INS-1 (832/13) cell line was kindly provided by Chris Newgard40 (link).

Tsend-Ayush E., He C., Myers M.A., Andrikopoulos S., Wong N., Sexton P.M., Wootten D., Forbes B.E, & Grutzner F. (2016). Monotreme glucagon-like peptide-1 in venom and gut: one gene – two very different functions. Scientific Reports, 6, 37744.

+ Open protocol

+ Expand

Calcium Imaging in 2D and 3D hCS Cultures

Check if the same lab product or an alternative is used in the 5 most similar protocols

For calcium imaging in monolayer, hCSs were dissociated and cultured for two weeks. Cells were loaded with 1 μM Fura-2 acetoxymethyl ester (Invitrogen) for 30 min at 37 °C in Neurobasal supplemented with B-27, washed with Tyrode’s solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose and 25 mM HEPES, pH 7.4) and placed in a perfusion chamber on the stage of an inverted fluorescence microscope (TE2000U; Nikon). Imaging was performed at room temperature (23–25 °C) on an epifluorescence microscope equipped with an excitation filter wheel and an automated stage. The Openlab software (PerkinElmer) was used to collect and quantify time-lapse excitation ratio images, and fluorescence images were analyzed with the IGOR Pro software (WaveMetrics). For calcium imaging in 3D cultures, intact hCSs were loaded with 1 μM Fluo-4 acetoxymethyl ester (Invitrogen) for 30 min at 37 °C in Neurobasal supplemented with B-27, washed and sliced in half. Live imaging was performed at the NMS (Stanford Neuroscience Microscopy Service, supported by a US National Institute of Health grant, NS069375) using a Zeiss LSM780 confocal microscope.

Paşca A.M., Sloan S.A., Clarke L.E., Tian Y., Makinson C.D., Huber N., Kim C.H., Park J.Y., O’Rourke N.A., Nguyen K.D., Smith S.J., Huguenard J.R., Geschwind D.H., Barres B.A, & Paşca S.P. (2015). Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods, 12(7), 671-678.

+ Open protocol

+ Expand

Monitoring Calcium Dynamics in MMSC-derived Cells

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

MMSC-derived cells were dissociated by incubation with collagenase and 0.25% trypsin for 10 mins. The dissociated cells were plated onto 25-mm microscope glass coverslips coated with growth factor-free Matrigel (diluted 1:60 in RPMI medium) and cultured overnight. Cells were then incubated with 5μM Fluo-4 acetoxymethyl ester (Invitrogen) in culture medium for 2 hrs. at 37°C. Using a standard Tyrode solution, a laser-scanning confocal microscope (Olympus) was used to measure the fluorescence intensity of Fluo-4 dye from the cells on a temperature-controlled plate (37°C). Cells with Fluo-4 dye were simultaneously excited at 340 (F340) and 380 nm (F380) and emission signals were collected at 505 nm by a photomultiplier tube. Changes of Fluo-4 fluorescence intensity (indicating transient fluctuation of cytosolic calcium concentration) were recorded in frame and line-scan mode as previously described [30 (link), 31 (link)]. Changes of intracellular Ca²⁺ content were expressed as changes in ratio ΔR = F340/F380. In addition, caffeine (Sigma) was directly added into the chamber that contained the beating MMSC-derived cells during imaging of calcium transient. Changes of Fluo-4 fluorescence intensity were confirmed and recorded with a confocal microscope.

Huang W., Liang J., Feng Y., Jia Z., Jiang L., Cai W., Paul C., Gu J.G., Stambrook P.J., Millard R.W., Zhu X.L., Zhu P, & Wang Y. (2018). Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential. Experimental cell research, 371(1), 20-30.

+ Open protocol

+ Expand

Intracellular Ca2+ Imaging of MSCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells in glass bottom dishes were loaded with Fluo-4 acetoxymethyl ester (Invitrogen, UK, 15μM, dissolved in DMSO with 0.1% pluronic F-127) for 15min at 24°C and then transferred to indicator-free solution for ≥30min. The dishes with Fluo-4 loaded cells were transferred to the stage of an inverted Olympus microscope. Superfusion of cells was performed by applying a positive pressure, valve-controlled, flow via a 100μm diameter tip attached to a 3-d mechanical manipulator (Narishige, Japan) which allowed positioning of the superfusion tip in the desired region of the dish. The solution was removed by suction at the other end of culture dish. All experiments were performed at 30°C. We used a Nipkow disc, confocal microscope (Ultraview, Perkin Elmer, Waltham, MA), connected to an iXon cooled charge-coupled device camera (Andor Technology, UK). Andor Technology iQ or iQ2 data acquisition software was used for 2- and 3-dimensional confocal imaging of Fluo-4 loaded MSCs. Images were collected at 33 frames per second using dry (×10, NA 0.42) or, water immersion (x40, NA 1.2) objectives (Olympus).

Kumar J.D., Holmberg C., Balabanova S., Borysova L., Burdyga T., Beynon R., Dockray G.J, & Varro A. (2015). Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF. PLoS ONE, 10(10), e0141331.

+ Open protocol

+ Expand

Fluo-4 Calcium Imaging in Cardiomyocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

The same samples were incubated with 3.5 µM Fluo-4-acetoxymethyl ester (Invitrogen, #F14201) in Tyrode´s solution (1.8 mM Ca²⁺) (1 hour, room temperature), washed (15 min) and transferred to a chamber containing parallel platinum electrodes. Fluorescence images were recorded after pacing (10 V, 0.5 msec) from CM showing clear striation and normal contractility. Sparks were analyzed using SparkMaster plugin (ImageJ).

Giudice J., Xia Z., Wang E.T., Scavuzzo M.A., Ward A.J., Kalsotra A., Wang W., Wehrens X.H., Burge C.B., Li W, & Cooper T.A. (2014). Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development. Nature communications, 5, 3603.

+ Open protocol

+ Expand

Calcium Mobilization and CD5 Expression Analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Calcium mobilization was also measured using flow cytometry and the high affinity calcium indicator Fluo-4 (ex:470–490 nm and em: 520–540 nm). Cells were surface stained with an anti-CD4⁺-APC antibody (17–0041; eBioscience). T cells were loaded for 30 mins as previously published with pluronic acid and 1mM Fluo-4-acetoxymethyl ester (Invitrogen) in Ringer solution (150 mM NaCl, 10 mM glucose, 5 mM of HEPES, 5 mM of KCl, 1 mM MgCl₂, and 2 mM CaCl₂, pH 7.4) [35 (link)]. Intracellular calcium mobilization was initiated by adding 50 ng/ml of PMA (phorbol 12-myristate 13-acetate) and 1 μg/mml of ionomycin [36 (link)]. For further analysis done in FlowJo, the lymphocyte population was gated in a forward and side scatter gate and singlets. From this gate a second gate was created specific for CD4⁺ T cells [37 (link)]. Intracellular calcium flux was measured in the CD4⁺ T cell gate using the FlowJo kinetics tool.
For CD5 expression analysis, spleen single cell suspensions from naïve and stimulated (day 3 and day 8) were stained with anti-CD5-PE (12–0051; eBioscience), and anti-CD4-APC (17–0041; eBioscience) and analyzed on the flow cytometer (BD Accurri C6).

Freitas C.M., Hamblin G.J., Raymond C.M, & Weber K.S. (2017). Naïve helper T cells with high CD5 expression have increased calcium signaling. PLoS ONE, 12(5), e0178799.

+ Open protocol

+ Expand

Calcium Signaling in HT-29/B6 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HT-29/B6 cells were seeded to the bottom of inverted filter supports (Millicell 12 mm; 0.4 μm pore size; Millipore; Germany) and imaging was performed on day 7 after confluency. Cells were washed once with HEPES/Ringer buffer (containing 2 g/l glucose, 2 mM probenecid, 1% penicillin/streptomycin). Subsequently, cells were loaded with the calcium dye Fluo-4-AM (4 mM Fluo-4-acetoxymethyl ester, Invitrogen) and the nucleus stain Hoechst 33342 (2 μM, Thermo Fisher Scientific, Waltham, MA., USA) for 1 h at 37 °C. For visualisation of cell borders 5 μg/ml CellMask plasma membrane stain (Invitrogen) was added. After washing thoroughly (3 × 5 min) with buffer, a first image was taken by confocal laser-scanning microscopy using a 40x water immersion objective (Zeiss LSM780, Jena, Germany) and maintained for 26 min with HlyA. After this first incubation period, images were taken every 2 min throughout the experiment with HlyA. The incubation chamber was heated to 37° and maintained with 5% CO₂ in ambient air throughout the experiment.

Wiegand S., Zakrzewski S.S., Eichner M., Schulz E., Günzel D., Pieper R., Rosenthal R., Barmeyer C., Bleich A., Dobrindt U., Schulzke J.D, & Bücker R. (2017). Zinc treatment is efficient against Escherichia coli α-haemolysin-induced intestinal leakage in mice. Scientific Reports, 7, 45649.

+ Open protocol

+ Expand

Laminarin Effects on Intracellular Calcium

Check if the same lab product or an alternative is used in the 5 most similar protocols

ES2 and OV90 cells were seeded in 6-well plates and incubated for 24 h in serum-free medium when the cells reached 70% to 80% confluency. The cells were then treated with different concentrations of laminarin for 48 h at 37 °C in a CO₂ incubator. Supernatants were discarded, and adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, resuspended in 3 μM fluo-4 acetoxymethyl ester (Invitrogen, Carlsbad, CA, USA), and incubated at 37 °C in a CO₂ incubator for 20 min. The stained cells were washed with PBS, and the fluorescence intensity was analyzed using a flow cytometer (BD Biosciences).

Bae H., Song G., Lee J.Y., Hong T., Chang M.J, & Lim W. (2020). Laminarin-Derived from Brown Algae Suppresses the Growth of Ovarian Cancer Cells via Mitochondrial Dysfunction and ER Stress. Marine Drugs, 18(3), 152.

+ Open protocol

+ Expand

Receptor Activation Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dulbecco’s modified Eagle’s medium (DMEM), hygromycin-B, and Fluo-4 acetoxymethyl ester were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Melbourne, Victoria, Australia). AlphaScreen™ reagents, and LANCE HTRF cAMP kit were purchased from PerkinElmer Life Sciences (Waltham, MA, USA). SureFire™ ERK1/2 reagents were generously supplied by TGR Biosciences (Adelaide, South Australia, Australia). GLP-1 was purchased from Mimotopes (Victoria, Australia).
All other reagents were purchased from Sigma (St. Louis, MO, USA) or BDH Merck (Melbourne, Vic, Australia) and were of an analytical grade.

Hager M.V., Clydesdale L., Gellman S.H., Sexton P.M, & Wootten D. (2017). Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1. Biochemical pharmacology, 136, 99-108.

+ Open protocol

+ Expand

Intracellular Ca2+ Signaling and IP Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols

Intracellular Ca²⁺ release was measured as described (Hlavackova et al., 2005 (link)). In brief, cells were pre-incubated for 1 hr with the Ca²⁺ -sensitive Fluo-4 acetoxymethyl ester (Invitrogen). The fluorescence signals (excitation at 485 nm and emission at 525 nm) were then measured for 60 s (Flex-Station, Molecular Devices). Agonist was added after the first 20 s. The Ca²⁺ response is given as the agonist-stimulated fluorescence increase. Concentration response curves were fitted using Graph Pad Prism.
Inositol phosphate (IP) accumulation in HEK293 cells co-transfected with indicated subunits was measured after stimulation with agonist for 30 min in 96-well microplates as previously described (Hlavackova et al., 2005 (link)). After incubation in the presence of LiCl (10 mM, 30 min) and termination of the reaction with 0.1 M formic acid, the supernatant was recovered and purified by ion exchange chromatography using DOWEX resin. Radioactivity was measured using a Wallac1450 MicroBeta microplate liquid scintillation counter (Perkin Elmer, Waltham, MA, USA).

Liu J., Zhang Z., Moreno-Delgado D., Dalton J.A., Rovira X., Trapero A., Goudet C., Llebaria A., Giraldo J., Yuan Q., Rondard P., Huang S., Liu J, & Pin J.P. (2017). Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer. eLife, 6, e26985.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!