The calcein am cell viability assay provides a simple, rapid, and accurate method to. This invention relates to a method for simultaneously detecting live and dead cells using two fluorogenic reagents. Direct assay or calcein am calcein am c3099 intracellular esterase activity indicates cell membrane integrity green fluorescence 1030 min 1. In addition, viability assays using calcein are reliable and correlate well with the standard crrelease assay. The assay is useful for various studies, such as cell viability, cell adhesion, chemotaxis, multidrug resistance, apoptosis and cytotoxicity. We standardized a microcytotoxicity test using calceinacetoxymethyl calceinam dye that requires very small quantities of cells while maintaining the same sensitivity as. 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 assay can be used for both suspension and adherent cells. Detergent and lysis buffer are provided for extracting the mtt reagent or the calcein amethd1 from cell samples. Calcein am is itself nonfluorescent and membranepermeant, and thus can be introduced into cells via incubation. Fluorometric cell introduction calcein am is a widely used green fluorescent cell marker. Cytotoxicity is one of the most important indicators for biological evaluation in vitro studies.
The hydrolysis of calcein am by intracellular esterases. Assay results obtained with this kit strongly correlate with the number of damaged cells. Overview calcein am cell viability assay kit fluorometric ab228556 is a simple, extremely sensitive quantitative assay to measure the viability of adherent and suspension cells that can detect as low as 50 viable cells in less than 30 minutes. Cytotoxicity assays provide an in vitro evaluation of the lytic activity of nk and t cells against tumors or transformed cells. An improved fluorescence assay for the determination of lymphocytemediated cytotoxicity using flow cytometry. Calcein am is a widely used green fluorescent cell marker. Tools for visualizing and quantifying neuronal cell health. In vitro, chemicals such as drugs and pesticides have different cytotoxicity mechanisms such as destruction of cell membranes, prevention of protein synthesis, irreversible binding to receptors etc. Test compounds and treatments were added for time periods appropriate to each assay. Trevigens calcein am cell viability kit provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity.
An improved flow cytometrybased natural killer cytotoxicity assay involving calcein am staining of effector cells. Livedead viabilitycytotoxicity kit thermo fisher scientific. Multiparametric analysis of cytotoxicity using cell. Calceinam is used as a cell viability stain and as a neutral substrate for multidrug mdr efflux transporters. The hydrolysis of calcein am by intracellular esterases produces calcein. We standardized a microcytotoxicity test using calcein acetoxymethyl calcein am dye that requires very small quantities of cells while maintaining the same sensitivity as the. Furthermore, there is a good correlation between results from the ldh cytotoxicity detection kit assay and the 51.
Calcein amconverting enzyme activity in the cytoplasm was evaluated by determining the median intensity of the calcein am signal. Assays and reagents for measuring cytotoxicity, proliferation. To evaluate the efficacy of the calceinam cytotoxicity assay, tests were performed in parallel to the 51cr standard procedure using the same target k562 and. The calcein am assay was also able to measure cytotoxicity against two adherent osteosarcoma cell lines, hos and saos2. Celigo demonstration experiment nk cellmediated cytotoxicity using calcein am 3 10021 rev a assay protocol and plate setup goal measure nk cellmediated cytotoxicity using calcein amstained k562 and imr32 for a duration of 4 hours. Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions.
Calcein am cell viability assay can be easily adapted to various fluorescence setups, such as microplate assays, fluorescence microscope and flow cytometry. Cytoselect cell viability and cytotoxicity assay kit. In live cells the nonfluorescent calcein am is converted to a greenfluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. Novel fluorescence assay using calceinam for the determination of human erythrocyte viability and aging daniela bratosin,1 laura mitrofan,2 carmen palii,3 jero. However, none of these methods allow the recovery of cells or supernatants after the assay. Cell counting kit8 uses a tetrazolium salt, wst8, which produces the water soluble wst8 formazan.
Calcein am is a nonfluorescent cell permeable derivative of calcein that becomes fluorescent upon hydrolysis within the cytosol. Assays to measure proliferation, viability and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The cytotoxicity is a very important aspect, as destruction of healthy living cells around the wound will have a negative impact on the healing process. The kit contains mtt reagent, calcein am, and ethidium homodimer. The high sensitivity of our assay is the result of a much higher accuracy of compound. Calcein does not inhibit any cellular functions such as proliferation or chemotaxis of lymophocyte. Measure nk cellmediated cytotoxicity using calcein amstained k562 and imr32 for a duration of 4. A flow cytometrybased cytotoxicity assay for the assessment. Author links open overlay panel xiu ming wang paul i. Instructions calcein am cell viability kit trevigen.
The enhanced hydrophobicity of the acetomethoxy am derivative of calcein allows this dye to readily enter viable cells. Cells were treated with hyaluronidase or vehicle pbs for 30 min, or tgf. We present a microtest for cellmediated immunity, based on the use of the tarasaki tray and calcein am vital dye. Calceinam solution should be used within 2 months after reconstitution and should be stored desiccated at 20c and protected from light. A new microcellular cytotoxicity test based on calcein am. Celigo demonstration experiment pbmcmediated cytotoxicity using calcein am 3 10027 rev a assay protocol and plate setup goal measure pbmcmediated cytotoxicity using calcein amstained k562 for a duration of 4 hours. Propidium iodide pi is membrane impermeant and therefore does not enter viable cells with intact membranes. Calcein am cell viability assay kit fluorometric nbp2. Celigo demonstration experiment nk cellmediated cytotoxicity using calcein am 3 10021 rev a assay protocol and plate setup goal measure nk cellmediated cytotoxicity using calcein am stained k562 and imr32 for a duration of 4 hours. Calcein am is membranepermeant and thus can be introduced into cells via incubation. Calcein am assay kit ab228556 is a simple, extremely sensitive quantitative assay to measure the cell viability of adherent and suspension cells. Among the dyes we have used, acetoxymethyl diacetylester of calcein calcein. 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. Each vial contains enough 4 mm calceinam stock solution for five 96well plates or 100 flow cytometry assays.
The livedead viabilitycytotoxicity assay kit provides a twocolor fluorescence. The calcein am cell viability assay provides a simple, rapid, and accurate method to measure cell viability andor cytotoxicity. Pdf a new microcellular cytotoxicity test based on calcein. In contrast, cells treated with 10 % dmso bottom result in small hoechst 33342stained nuclei and only a small number of cells that are positive for calcein am. In our hands, the calcein release assay and the ldh release assay yielded an ec50 of 3. The calceinam assay was also able to measure cytotoxicity against two adherent osteosarcoma cell lines, hos and saos2. Cell counting kit8 product description cell counting kit8 is a colorimetric assay for the determination of viable cell numbers and can be used for cell proliferation assays as well as cytotoxicity assays.
Calceinam is the most suitable fluorescent probe for staining viable cells because of its low cytotoxicity. Calcein am is a nonfluorescent, hydrophobic compound that easily penetrates intact and live cells. Calcein am was purchased from invitrogenmolecular probes carlsbad, ca and used at a concentration of 125 nm for analysis by flow cytometry, and 2. Once inside the cells, calcein am, a nonfluorescent molecule itself, is hydrolyzed by endogenous esterases into the highly negatively charged green fluorescent calcein. Maximum and spontaneousrelease values were, respectively, 5,854 1,0 and 1,849 277 afu for hos cells and 5,292 345 and 1,485 68 afu for saos2. Calceinacetyoxymethyl cytotoxicity assay clinical and vaccine. Am, a derivative of fluorescein, stands out as the first indicator of cell viability and cellular. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. K562 cells were obtained from atcc and cultured in rpmi 1640 media 2.
Calcein am structure a is a nonfluorescent, hydrophilic compound that easily permeates intact, live cells. Fluorometric cell viability kit ii calcein promocell. Cellbased assays using calcein acetoxymethyl ester show. Ethd1, the deadcell indicator in our livedead viabilitycytotoxicity kit l3224, viability and cytotoxicity assay kits for diverse cell typessection 15. The assay monitors the inhibition of the mdr1pgp abcb1 or mrp1 abcc1 mediated efflux of calcein am by measuring the increased fluorescence of the calcein dye. K562 and imr32 were obtained from atcc and cultured in rpmi 1640 media 2. Calcein am is a cellpermeant dye that can be used to determine cell viability in most eukaryotic cells. Cell biolabs cytoselect cell viability and cytotoxicity assay kit provide a colorimetric and fluorometric format for measuring and monitoring cell viability. Key benefits suitable for proliferating and nonproliferating cells. The calcein am dye used to stain the living cells was shown to have a low.
Cell biolabs cytoselect cell viability and cytotoxicity assay kit provides a colorimetric and fluorometric format for measuring and monitoring cell viability. Calcein am is used as a cell viability stain and as a neutral substrate for multidrug mdr efflux transporters. Introduction trevigens calcein am cell viability kit provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity. Any tips for a cytotoxicity assay by calcein am labeling. This dye is also available as 1 mg of the solid c1430 and resuspended in dmso c3099. 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. Quantification of number of viable cells is an indispensible tool in cell biology research. The calcein am cell viability assay provides a simple, rapid and accurate method to measure cell viability andor cytotoxicity. 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. I cant seem to find a video performing this part of the procedure.
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. In order to determine the cell death caused by these damages, there is a need for cheap, reliable. Hydrolysis of calcien am dye to fluorescent calcein. The number of target cells needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Calcien am dye is susceptible to hydrolysis and so the working solution should be used within 24 hrs after preparation. Us5314805a dualfluorescence cell viability assay using. Nk cellmediated cytotoxicity using calcein am nexcelom bioscience. Viability and cytotoxicity assay reagentssection 15. Pdf cytotoxicity assays provide an in vitro evaluation of the lytic activity of nk and t cells. 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.
Calcein, am, cellpermeant dye thermo fisher scientific. Out application allows to make a quick, fully automated evaluation of livedeath cells. Calcein am cytotoxicity assays based on the evaluation of dye released in the supernatant used a relatively high number of target cells 15. Live cells can convert the nonfluorescent, cellpermeable polyanionic calcein. It can detect as low as 50 viable cells in less than 30 minutes. For fluorescence microplate reader, fluorescence microscopy, or flow cytometry.
Calcein am is a nonfluorescent, hydrophobic compound that easily permeates intact, live cells. Im doing a cytotoxic assay by labeling my target cells with calcein am, cocultured with. 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. Live cells are distinguished by an intense uniform green fluorescence generated by the enzymatic hydrolysis of calcein am. In addition, viability assays using calcein are reliable and correlate well with the standard.
1093 351 808 1261 1450 1395 1429 446 966 144 1471 57 98 1188 1294 156 1555 410 674 347 1555 1194 235 576 753 965 138 418 1233 376 279 422 1456 1082 1365 316 455 497 284 1122 617 1351