To define an assay that is optimal for flow cytometric detection of mrp1 functional activity in clinical samples, we used both of these am dyes as well as the pgp. And the results can be obtained quickly, mostly within 2 hours. Nov 16, 2004 however a practical assay to accurately determine the pump function of p gp is still lacking. The loss of the acetoxymethyl group also enables calcein to readily bind intracellular calcium resulting in a stro. When the acetoxymethyl ester is intact, this probe is nonfluorescent until acted upon by nonspecific esterases present within the healthy, live cells. Mitochondria function assays thermo fisher scientific uk. Cells were treated with hyaluronidase or vehicle pbs for 30 min, or tgf. Cells are loaded with calcein am, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Comparison of 3 assay systems using a common probe substrate, calcein am, for studying p gp using a selected set of compounds peter szeremy, akos pal, dora mehn, beata toth, ferenc fulop, peter krajcsi, krisztina herediszabo, 2011. Once inside the cells, endogenous esterases hydrolyze the compound into the highly negatively charged green fluorescent dye calcein, which is retained in the cytoplasm in. Calcein am cell viability assay can be easily adapted to various fluorescence setups such as microplate assays, fluorescence microscope and flow cytometry.
Differences in the automatic fluorescence readings between the test and control wells determine the results1. The fluorescent signal generated from the assay is proportional to the number of living cells in the sample. The calcein am cell viability assay kit is a fluorometric method for extremely sensitive quantification of viable cells that can detect as few as 50 viable cells in less than 30 min. Viability measurement of a549 cells labeled with calcein am, propidium iodide and hoechst calcein am detects enzymatic activity in live cells. Prepare 1 mm calceinam solution with dmso and dilute to prepare 150. Propidium iodide pi is membrane impermeant and therefore does not enter viable cells with intact membranes. Calceinam for determination of cell vitality nexcelom. Quantification of number of viable cells is an indispensible tool in cell biology research. Viability staining protocol calcein am and propidium iodide.
To overcome this issue, we decided to carry out mtt assays, to take into. Calcein am has been used in a retention assay for this purpose, whereas bcecf am has been used in an accumulation assay. Pglycoproteinactivity measurements in multidrug resistant. This calceinam assay should open the way for ranking large numbers of novel structures for their potential pgp modulator properties, particularly for an efficient screening of pgp function antagonists, but it does not allow defining whether their inhibition may be competitive or not. Return the unused portion of the calcein am stock solution to storage at 20 c under desiccation. Calcein am is a nonfluorescent, hydrophobic compound that easily penetrates intact and live cells. Acquire a wholewell image of an entire 96well plate on celigo 15 minutes results. Vybrant multidrug resistance assay kit thermo fisher scientific. Solvos patented calcein assay is an indirect inhibitorytype whole cell assay that provides information on any interaction between the abc transporter and the test drug. Supplied in lyophilized form or as a solution in anhydrous dmso at 4 mm 800111 or 1 mgml 1 mm 800112. Non fluorescent calcein am cam diffuses passively through the plasma.
Using a common probe allows the investiga tion of the effect of passive permeability on the result obtained by testing various compounds. Im doing a cytotoxic assay by labeling my target cells with calcein am, cocultured with different ratios of effector cells and checked the calcein am released by lysed cells. Key benefits suitable for proliferating and nonproliferating cells. A transported substrate was positive in the efflux. The calcein assay is based on the conversion of the cell permeant nonfluorescnt calcein am dye to the fluorescent calcein dye by intracellular esterase activity in live cells. Novel fluorescence assay using calceinam for the determination of human erythrocyte viability and aging. The use of calcein acetomethylester amlabelled polymorphonuclear cells in a polycarbonate filter chemotaxis assay. Calceinam is a nonfluorescent lipophilic ester that easily penetrates cellular membranes. The vybrant multidrug resistance assay kit provides a rapid and simple method for largescale screening of mdr inhibitors. This probe substrate is calcein acetoxymethyl ester calcein am, the acetoxymethyl ester derivative of the fluorescent dye, calcein. Calceinam cas 148504341 calbiochem 1 product result match criteria.
Calcein am is a cellpermeable, nonfluorescent acetoxymethyl ester derivative of calcein that gets hydrolyzed inside the cell by esterases. Multidrug resistance assay kit calcein am cayman chemical. In this study, an assay based on a fluorescent dye calcein acetoxymethyl esteror, calceinam and multiparameter flow cytometry capable of accurately determining the pump function was established. The efflux assay is more reliable at lowmoderate p app and is the method of choice for evaluating drug candidates despite low throughput and reliance on liquid chromatography with tandem mass spectrometry.
This dye is also available as 1 mg of the solid c1430 and resuspended in dmso c3099. Calcein, am, cellpermeant dye from thermo fisher scientific. Pgp was the first drug efflux discovered and attributed to multidrug. Invitrogen calcein, am, cellpermeant dye 20 x 50ug. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. Calcein am cell viability assay is more robust than tetrazolium salt or alamarblue dye as the cells can be stained and quantified in less than 2 hrs. On the other hand, presence of pgp inhibitor increases cellular entry of calcein am and thereby increases cellular fluorescence. Calcein am is used as a cell viability stain and as a neutral substrate for multidrug mdr efflux transporters. Calcein, am ultrapure grade cas 148504341 aat bioquest. Will gfp interfere with calcein fluorescence during an. Any tips for a cytotoxicity assay by calcein am labeling. Comparison of 3 assay systems using a common probe. In metabolically active cells, calcein am is converted by cytosolic esterases into green fluorescent calcein. Pglycoprotein 1 pgp is a cell membrane protein that functions as an atpdependent drug efflux pump and plays an important role in multidrug resistance.
Calcein assay solvos patented calcein assay provides information on the interaction between mdr1 pgp abcb1 or mrp1 abcc1 transporters and the test compound. Calcein am or calcein acetoxymethyl ester is a hydrophobic compound, which passes easily through cell membranes into live cells and is used for cell viability assays. The calcein am cell viability assay provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity. Immediately prior to use, dilute the calcein am stock solution in 1x calcein am dw buffer to a 2x calcein am working solution, preparing enough for all wells using 50. Marie, pgp and mrp activities using calceinam are prognostic.
In addition, it can be performed in a barrierspecific manner highlighting the dependence of abcb1 ic50 values on. Calcein am is itself nonfluorescent and membranepermeant, and thus can be introduced into cells via incubation. One assay to confirm expression pattern of pgp is the calcein. The calcein am cell viability assay provides a simple, rapid, and accurate method to measure cell viability andor cytotoxicity. Calceinam uptake and efflux have been studied in cell lines, but not in fresh cells of aml patients, except for one publication with few patients studied 14 patients for calceinam. Rational use of in vitro pglycoprotein assays in drug.
The calcein am cell viability assay is an endpoint analysis method for cell viability. Apr 15, 2007 the cytochemical calceinam calceinacetoxymethyl ester method 14 is an established technique for the assay of the cellular labile iron pool lip. The hydrolysis of calcein am by intracellular esterases. Nonfluorescent, hydrophobic calcein am dissolved in the lipid bilayer is pumped out of the cell by mdr1pgp abcb1 or mrp1 abcc1, thus keeping the. Cells were seeded at 50,000 cellswell in a 96well plate and incubated for 24 h. Calcein, am, cellpermeant dye,cell viability genecopoeia.
The assay monitors the inhibition of the mdr1p gp abcb1 or mrp1 abcc1 mediated efflux of calcein am by measuring the increased fluorescence of the calcein dye. This assay uses the fluorogenic dye calcein, am as a substrate for efflux activity of pgp. This assay employs the acetoxymethyl am ester of calcein, a colorless and nonfluorescent esterase substrate, and cocl 2 cobalt, a quencher of calcein fluorescence, to selectively label mitochondria. Am was prepared as a stock solution of 10 mm in dimethylsulfoxide stored at. Am is a substrate of pgp and emits no fluorescent signal itself. Establishment of optimized mdck cell lines for reliable. Calcein am structure a is a nonfluorescent, hydrophilic compound that easily permeates intact, live cells. I am doing an invasion assay that uses calcein am but the cells i want to assay contain the gfp gene and since they emit at approximately the same wavelength, i am not sure if it is feasible to. Pgp, the product of the multidrug transporter mdr1 gene, actively extrudes the calceinam, but not the fluorescent calcein.
Calcein am is a nonfluorescent cell permeable derivative of calcein that becomes fluorescent upon hydrolysis within the cytosol. The hydrolysis of calcein am by intracellular esterases produces calcein structure b, a hydrophilic, strongly fluorescent compound that is wellretained in the cell cytoplasm. This dye is provided as 1 mg solid c025 and 1 mgml solution in dmso c026. The enhanced hydrophobicity of the acetomethoxy am derivative of calcein allows this dye to readily enter viable cells. Calcein am cell viability assay kit fluorometric nbp2. Using this software, a segmentation mask was generated to separate. Multidrug resistance reversal agent, nsc77037, identified. Calcein am calcein acetoxymethyl ester is a nonfluorescent compound that passively enters cells. The high sensitivity of our assay is the result of a much higher accuracy of compound. In our hands, the calcein release assay and the ldh release assay yielded an ec50 of 3. The flow cytometrical evaluation of pgp pump function on. The fluorescent calcein is retained by live cells with intact membranes. Calcein am is a widely used green fluorescent cell marker.
Cells were treated with either nsc77037 or verapamil for 1 h and then incubated with calcein am for 30 min. Calcein am is a cellpermeant dye that can be used to determine cell viability in most eukaryotic cells. The calcein assay is a highthroughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in mdckiimdr1 cells. Calcein am is also a substrate for pglycoprotein pgp. Mdamb231 were obtained from atcc and cultured in rpmi 1640 media 2. Biovisions calcein am cell viability assay kit is a fluorometric method for extremely sensitive quantification of viable cells that can detect as low as 50 viable cells in less than 30 min.
Calceinam is a fluorogenic, cellpermeant fluorescent probe that indicates cellular health. Celigo demonstration experiment nk cellmediated adcc using calcein am 3 10024 rev a assay protocol and plate setup goal measure adcc of nk92 cellmediated cytotoxicity using calcein amstained mdamb231 for 6 hours protocol cell preparation 1. The assay detects both pglycoprotein and multidrug resistanceassociated protein activities, and identifies aml patients with unfavourable therapy responses. Nonfluorescent, hydrophobic calcein am dissolved in the lipid bilayer is pumped out of the cell by mdr1 pgp abcb1 or mrp1 abcc1, thus keeping the intracellular concentration of calcein am low. Calcein am is nonfluorescent, highly lipidsoluble dye that can rapidly penetrate the plasma membrane of normal cells. At the end of the reaction, unmetabolised atp is detected as a luciferasegenerated luminescent signal. The hydrolysis of calcein am by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that is wellretained in the cell cytoplasm. For the calceinam efflux assay, cells were cultured at 80 or 90% confluency in t25. The calcein am assay was optimized and performed using the vybrant multidrug resistance kit. Pglycoproteinactivity measurements in multidrug resistant cell. Introduction trevigens calcein am cell viability kit provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity.
Ranking of pglycoprotein substrates and inhibitors by a. In live cells the nonfluorescent calcein am is converted to a greenfluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. Invitrogen calcein, am, cellpermeant dye chemicals. Am assay, which measures the intracellular accumulation of fluorescent calcein. There are however, as we shall demonstrate, reasons to doubt its efficacy. Probes for cell adhesion, chemotaxis, multidrug resistance.
Flow cytometric assay of cell viability using calcein. Dye efflux assays calcein assay, hoechst assay technologies. The assay is useful for various studies, such as cell viability, cell adhesion, chemotaxis, multidrug resistance, apoptosis and cytotoxicity. In this assay, recombinant human pgp is incubated in the presence of compounds and atp. Only cells possessing active cytosolic esterases fluoresce green. Calcein am can be purchased from invitrogen ebioscience, product number 15560597. Calcein am cell viability assay can be easily adapted to various fluorescence setups, such as microplate assays, fluorescence microscope and flow cytometry.
Test compounds and treatments were added for time periods appropriate to each assay. These include monolayers of caco2 cells, which express native pgp, other cell lines. Cellbased assays using calcein acetoxymethyl ester show. The experiments were initiated by washing the cells with pbs. A simple and sensitive cellcell adhesion microplate assay is established. We conclude that calcein am, when used in a retention assay with mrp1specific modulators, is able to reliably. Dec 26, 2002 calcein am has been used in a retention assay for this purpose, whereas bcecf am has been used in an accumulation assay. Atpase assays revealed pgp interactions for highly permeable group iia. In live cells the nonfluorescent calcein am is converted to a greenfluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. Rational use of in vitro pglycoprotein assays in drug discovery. Pgp and mrp activities using calceinam are prognostic. This point may not be relevant to drug screening programs carried out in. Calcein assay for multidrug resistance reliably predicts. Invitrogen calcein, am, cellpermeant dye 20 x 50ug chemicals.
Calceinam is widely used to quantify mdr efflux activity, as this molecule behaves as. Comparison of 3 assay systems using a common probe substrate. This assay utilizes the fluorogenic dye calcein am as a substrate for efflux activity of pgp. In this study, a common probe substrate was applied in 3 assay systems developed to study mdr1. Calcein am is a nonfluorescent, hydrophobic compound that easily permeates intact, live cells.
Upon hydrolysis by intracellular esterases, calcein is well retained in the cytosol and, unlike the hydrolysis product of other fluorescent pgp substrates such as bcecf am or fura2 am, its fluorescence is neither ph nor calcium dependent. Experimental procedures definitions of nonsubstrate, inhibitor, and substrate a nonsubstrate was negative in all three assays table 2. An inhibitor was positive only in the calceinam assay. Calcein am plays an important role in early drug discovery. Calcein am is an excellent tool for the studies of cell membrane integrity and for cell tracing. Presence of pgp results in efflux of calcein am and thereby decreases cellular fluorescence. Cleaving the am ester allows the probe to excite and emit at 488nm 520nm respectively.
A highthroughput assay for measuring abcb1mediated calcein am efflux. Calcein am is nonfluorescent, highly lipidsoluble dye that can rapidly. Calcein assay license product categories solvo biotechnology. Calcein am is an organic heteropentacyclic compound that is calcein in which all four carboxy group hydrogens have been substituted by acetyloxymethoxy groups and the hyrodgens of the two hydroxy groups have been substituted by acetyl groups. Calcein am is also offered in our cell viability and cytotoxicity assay kit. Unused portions of the dmso stock can be aliquoted and stored.
When p app is low, efflux is a greater factor in the disposition of pgp substrates. It is a a nonfluorescent probe cleaved to a fluorescent probe by nonspecific intracellular esterases. The filtermirror combination used to detect calceinams green fluorescence includes the 490nm excitation and 520nm emission filters with a dichroic mirror. The fluorescent calcein is wellretained in the cytoplasm in live cells. The assay is useful for various studies, such as cell viability, cell adhesion, chemotaxis, multidrug resistance, apoptosis and. Nonfluorescent cell permeable derivative of calcein, becomes fluorescent on hydrolysis. Calcein, am, cellpermeant dye thermo fisher scientific.
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