Celltrace calcein redorange am is a cellpermeant dye that can be used to determine cell viability in most eukaryotic cells. Calcein am has also been tested on mouse data not shown. Cell staining should be optimal at 15 x 105 cellswell, however this may vary with cell type. Cells grown in blackwalled plates can be stained and quantified in less than two hours. Calceinam, acetoxymethyl ester of calcein, is highly lipophilic and cell membrane. Calcein am, fluorescent dye for cell viability cas 148504341, with 98% purity. 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. Promokines livedead cell staining kit ii provides a twocolor fluorescent staining of live green and dead cells red using two probes and is suited for animal live and dead cells. Staining procedure for a fluorescence microscopy for 24well plate. The enhanced hydrophobicity of the acetomethoxy am derivative of calcein allows this dye to readily enter viable cells.
It has been used to study cytotoxic effects of drugs in cells. Calcein am is an excellent tool for the studies of cell membrane integrity and for cell tracing. Create a fresh aliquot of 500x calcein am solution in pbs from. Calcein am 4mmdmso,90%hplc,solution 148504341 sigma. Wash cells twice with pbs or an appropriate buffer. Instructions calcein am cell viability kit trevigen. The calcein am cell viability assay provides a simple, rapid, and accurate method to measure cell viability andor cytotoxicity.
I understand the staining procedure, but its the wellplate reader results that are confusing me. Ethidium bromide staining for analysis of cell death. Staining solution should be used within 2 hours from preparation. Once i stain the cells, incubate for an appropriate amount of. It can detect as low as 50 viable cells in less than 30 minutes. Due to a timeconsuming experimental setup, it would be very helpful if the cells could be fixated after calcein staining. Cell viability analysis using calcein am propidium iodide. Calcein am is itself nonfluorescent and membranepermeant, and thus can be introduced into cells via incubation. Store the stock of calcein am in the freezer directly after use. Calcein am was purchased from invitrogenmolecular probes carlsbad, ca and used at a concentration of 125 nm for analysis by flow cytometry, and 2. Images acquired every 3 seconds and total elapsed time on video is. Confident assurance using calcein am with minimal effect. Calcein am is a cellpermeant dye that can be used to determine cell viability in most eukaryotic cells.
Calceinam is the most suitable fluorescent probe for staining viable cells because of its low. Images were obtained using a light microscope bx51, olympus with fluorescent light source and. Prepare 1 mm calceinam solution with dmso and dilute to prepare 150. Target cell viability was not affected by calceinam staining, as assessed by the eosin exclusion test data not shown. These probes measure two recognized parameters of cell viability. Does anyone have experience with fixation of cells before. However, none of these methods allow the recovery of cells or supernatants after the assay.
This was originally used to measure ph inside the cell, and is also used as a dye to stain living cells. Calcein am, acetoxymethyl ester of calcein, is highly lipophilic and cell membrane. Cytotoxicity assays provide an in vitro evaluation of the lytic activity of nk and t cells against tumors or transformed cells. Tools for visualizing and quantifying neuronal cell health. Calcein am is the acetomethoxy form of calcein, a highly lipophilic, cell membrane permeable dye. Calcein am is a nonfluorescent, hydrophilic compound that easily permeates intact, live cells. Celltrace calcein redorange am is a cell permeant dye that can be used to determine cell viability in most eukaryotic cells. Adherent cells may be stained in suspension or in situ.
In cells, calcein am solution has been used in calceinam dye transfer assay. It has been used for the staining of cells to study quantification of cellmigration. The hydrolysis of calcein am by intracellular esterases produces calcein structure b, a hydrophilic, strongly fluorescent compound that is wellretained in the cell cytoplasm. The kit contains calceinam, which stains viable cells, and propidium iodide pi, an indicator of dead cells. How come calcein positive cells are also pi positive.
Us5314805a dualfluorescence cell viability assay using. The best concentration for calceinam and pi depends on the cell type, so it is necessary to determine the concetration when staining each cell. It is also used traditionally as a complexometric indicator for titration of calcium ions with. Calcein am is also offered in our cell viability and cytotoxicity assay kit.
Calcein am assay kit ab228556 is a simple, extremely sensitive quantitative assay to measure the cell viability of adherent and suspension cells. Thaw one calcein am vial 50 g and add 30 l of tissue culture grade dmso to prepare a working solution. In theory, dapi blue allows us to locate all cells, calcein am green indicates viability and propidium iodide red. The hydrolysis of calcein am by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that is wellretained in the cell cytoplasm. The mean spontaneous releases from k562 cells were about 10 and about 34% of the maximum for 51cr and calceinam assays, respectively. We standardized a microcytotoxicity test using calceinacetoxymethyl calceinam dye that requires very small quantities of cells while maintaining the same sensitivity as. Calcein am stains live cells green, while ethdiii stains dead cells red. This dye is also available as 1 mg of the solid c1430 and resuspended in dmso c3099. Calcein, also known as fluorexon, fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495515 nm, respectively, and has the appearance of orange crystals.
Invitrogen calcein, am, cellpermeant dye 20 x 50ug. Calceinam is cell membrane permeable and the generated calcein by esterase activity in the viable. Calcein am is a cell permeant dye that can be used to determine cell viability in most eukaryotic cells. This is a suggested procedure, please adjust according to your. Supplied in lyophilized form or as a solution in anhydrous dmso at 4 mm 800111 or 1 mgml 1 mm 800112. To further confirm that the higher spontaneous release with calceinam than with 51 cr was not due to a poor dyedependent target cell viability, a double labeling of k562 cells, first with calceinam and subsequently with 51. Cellstaindouble staining kit consists of two staining compounds, calceinam and propidium iodide pi.
Calcein blue is optimally excited at the 360 nm wavelength of light that can be provided by an ultraviolet laser and emits maximally at 445 nm. Prepare a staining solution of 2 m calceinam4 m ethdiii by adding 5 l of 4 mm calceinam and 20 l of 2 mm ethdiii to 10 ml of pbs or other serumfree buffer or medium. Invitrogen calcein, am, cellpermeant dye 20 x 50ug chemicals. Cells cultured in blackwalled plates can be stained and quantified in less than two hours. The hydrolysis of calcein am by intracellular esterases. M ethdiii in phosphatebuffered saline pbs was added to each membrane and incubated with cells for 3045 min at room temperature. Upon crossing the cell membrane, calceinam is rapidly hydrolyzed by cellular esterases inside live cells. Calcein am structure a is a nonfluorescent, hydrophilic compound that easily permeates intact, live cells. The hydrolysis of calcein am by intracellular esterases produces calcein. Intracellular esterase converts the nonfluorescent calcein am to the highly fluorescent calcein. Intracellular esterase converts the nonfluorescent calceinam to the highly fluorescent calcein. After entering into a cell, it combines with the amino base of protein in the cell membrane on the. Cellbased assays using calcein acetoxymethyl ester show.
Calcein am calcein acetoxymethyl ester is a cell permeable, nonfluorescent compound. Cam cell staining are 40% smaller in size compared with labelfree and lower cam cell staining conditions. Cell staining reagents target dye excitation emission excitation filtercolor characteristic bcecfam 490 nm 526 nm b excitation yellowish green calceinam 490 nm 515 nm b excitation yellowish green cfse 496 nm 516 nm b excitation yellowish green cytored 535 nm 590 nm g excitation red. Nonfluorescent cellpermeant calceinam enters the cell and is cleaved by ubiquitous esterases in living cells, producing. Calcein am, calcein violet am, and calcein blue am labeling dyes cross the cell membrane easily, selectively labeling live cells for analysis by flow cytometry or fluorescent microscopy. Prewarm staining solutions to 37c immediately before staining.
Join researchers using our high quality biochemicals. Target cell viability was not affected by calcein am staining, as assessed by the eosin exclusion test data not shown. Calcein am cell viability assay can be easily adapted to various fluorescence setups, such as microplate assays, fluorescence microscope and flow cytometry. What is the correct protocol for a livedead assay in a. This dye is provided as 1 mg solid c025 and 1 mgml solution in dmso c026. Indeed the calcein am is a test to reveal live cells. This kit contains calcein am and propidium iodide pi solutions, which stain viable and dead cells, respectively. We are staining our endothelial cells with the live cell staining calcein. Calcein am cell viability assay is more robust than tetrazolium salts or alamarblue dye, as the cells can be stained and quantified in less than 2 hrs. Livedead viabilitycytotoxicity kit thermo fisher scientific.
Cell staining reagents target dye excitation emission excitation filtercolor characteristic bcecf am 490 nm 526 nm b excitation yellowish green calcein am 490 nm 515 nm b excitation yellowish green cfse 496 nm 516 nm b excitation yellowish green cytored 535 nm 590 nm g excitation red. Cell viability analysis using calcein am propidium iodide and. Unlike calcein am c1430, c3099, c3100, celltrace calcein redorange am is intrinsically fluorescent. We are developing an assay to measure an analogue for cell transfection.
Cells were thawed and stained with bd calcein am cat. The assay is useful for various studies, such as cell viability, cell adhesion, chemotaxis, multidrug resistance, apoptosis and cytotoxicity. The best concentration can be determined using the following protocol. Cell counting viability reagents nexcelom bioscience. Calcein violet am is an optimal dye for this application. Calcein am is a pgp substrate and is effluxed from the cell left, forming minimal fluorescent calcein. Bd pharmingen calcein am is used for labeling live cells that can be detected and further analyzed by flow cytometry or fluorescence imaging. L of the combined livedead cell staining solution 2. Live cells are distinguished by an intense uniform green fluorescence generated by the enzymatic hydrolysis of calcein am. Using the cell counts, comparison of unlabeled and fluorescently labeled macrophages demonstrated that bcecf am decreased the number of cells responding to zas, while calcein am had essentially no effect. To further confirm that the higher spontaneous release with calcein am than with 51 cr was not due to a poor dyedependent target cell viability, a double labeling of k562 cells, first with calcein am and subsequently with 51. Cell staining protocol for fluorescence microscopy. To stain adherent cells in suspension, follow the general procedure as outlined above after cell.
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. Jul 17, 2009 mcf7 cells grown in 8well slide chamber for 2 days. The fluorescent chromophore, calcein c 30 h 26 n 2 o, specifically binds to calcium, fluorescently staining the calcified skeletal structures in living zebrafish larvae and juveniles fig. Data was acquired on a bd accuri c6 flow cytometer with or without the use of an fl1 90% attenuation filter cat. Calcein am is a nonfluorescent, hydrophobic compound that easily permeates intact, live cells. Trevigens calcein am cell viability kit provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity. Cell cytosol staining fluorogenic esterase substrates that can be passively loaded into viable cells, such as calceinam, bcecfam, carboxyfluorescein succinimidyl ester cfse, and fluorescein diacetate fda, are converted by intracellular esterases into fluorescein analogs with green fluorescence. Calceinam is a widely used membranepermeant cell marker that readily passes through the cell membrane of. Fluorescent staining of human u2 os cells with bd pharmingen calcein am. The calcein am staining must to be performed in live cells, otherwise you will never have the staining. Calcein am is a nonfluorescent, hydrophobic compound that easily penetrates intact and live cells. This kit is utilized for a simultaneous detection of viable cells and dead cells in a cell culture dish by using a fluorescent microscope. Viability assessment of fresh and frozenthawed isolated. Since dead cells lack esterase activity, only viable cells are labeled.
Calceinam is the acetomethoxy form of calcein, a highly lipophilic, cell membrane permeable dye. In live cells the nonfluorescent calcein am is converted to a greenfluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. Introduction trevigens calcein am cell viability kit provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity. Propidium iodide pi is membrane impermeant and therefore does not enter viable cells with intact membranes. In live cells the nonfluorescent calcein am is converted to a greenfluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. In the presence of a pgp inhibitor medium gray hexagon, the efflux of calcein am is reduced and more fluorescent calcein is produced right. Viability staining protocol calcein am and propidium iodide. Standard protocol for calcein am staining of living cells. Add calceinam solution with 110 of the volume of cell culture medium to the cell culture. Cells from the u2 os osteosarcoma, atcc htb96 cell line were stained with 10. For all experiments involving calcein labeling, cells were incubated with calcein am for 30 min followed by washing with pbs before subsequent analysis or procedures. Calcein, am, cellpermeant dye thermo fisher scientific. The kit contains calcein am, which stains viable cells, and propidium iodide pi, an indicator of dead cells. Calcein am is a widely used green fluorescent cell marker.
The best concentration for calcein am and pi depends on the cell type, so it is necessary to determine the concetration when staining each cell. This kit contains calceinam and propidium iodide pi solutions, which stain viable and dead cells, respectively. E quantification of single colony wells derived from single cells shows consistent data to raw outgrowth percentages outlined in b. Cell migration, chemotaxis and invasion assay protocol. Calcein selfquenches at concentrations above 70mm and is commonly used as an indicator of lipid vesicle leakage. Combine 1x cds with calcein am solution at a ratio of 1. Prepare 1x cell dissociation solution cds by diluting 10x cell dissociation solution with sterile filter deionized water. 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. If desired, 5 m calcein am molecular probes can also be added to counterstain viable. Staining is fast and simple, requiring only 30 minutes.
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