GE Healthcare Application Note 28-4070-51 AA IN Cell Analyzer 3000 Performance of cell viability and cytotoxicity assays on the IN Cell Analyzer 3000 Key words: cell-based assay viability cytotoxicity cytostatic IN Cell Analyzer Fluorescent dyes that indicate cellular viability through changes in intensity are used in many areas of cell biology to provide the effective cytotoxic dose of a variety of agents and processes including small molecules, overexpressed endogenous proteins, bacterial and viral toxins, and cell-mediated cytotoxicity. In addition, the use of fluorescent cytotoxicity assays as an initial step in compound screening facilitates more effective and relevant subsequent assays by highlighting sub-toxic concentrations of compounds within a library. Fluorescent cell viability assays can be performed on fluorescence plate readers, flow cytometers, or confocal and epifluorescence microscopes. Flow cytometers and fluorescence microscopes provide information on viability through assessment of individual cell fluorescence and permit correlation of viability, phenotype, and cell number. However, flow cytometers and conventional fluorescence microscopes have limited throughput capability. Fluorescence plate readers provide throughput but report only an indication of cell viability within a well through the assessment of total well fluorescence and this value cannot be easily related to the exact cell number or phenotype. High-throughput fluorescence microscopic imaging and analysis combines the throughput obtained using plate readers with the information content obtained using flow cytometers and conventional fluorescence microscopes to provide a high-content, high-speed solution. This application note describes the high-speed acquisition of images from cell viability assays using the IN Cell Analyzer 3000, and provides methods for analysis using the IN Cell Analyzer 3000 Cell Viability Analysis Module. Materials Products used IN Cell Analyzer 3000 25-8010-11 IN Cell Analyzer 3000 Seat License* 63-0055-97 Other materials used HeLa, U-2 OS, A549, and SH-SY5Y cells (ATCC) DMEM supplemented with 10% fetal bovine serum, 2-mM L-glutamine, 100-µg/ml penicillin, and 100-µg/ml streptomycin McCoys 5A medium with supplements as for HeLa cells SH-SY5Y cells 1:1 mixture of HAM F12 and Minimal Essential Medium Eagle with the supplements as for HeLa cells and additional 1 non-essential amino acids HAM F12 with supplements as for HeLa cells Ionomycin Vinblastine Paclitaxel Staurosporin Calcein-AM Propidium iodide Hoechst 33342 96-well Viewplates (Perkin Elmer) * A seat license is a cost-effective single-user or server license that gives access to all ready to use Image Analysis Modules provided for your IN Cell Analyzer instrument. License holders have access to all appropriate analysis software and more licenses can be purchased as the number of users grows.
Method The cell viability assay described here involves the simultaneous application of three fluorescent dyes directly to cells in culture medium, subsequent live-cell image acquisition, and analysis on the IN Cell Analyzer 3000 (Fig 1). Hoechst 33342 is used as a blue fluorescent marker of the nuclei of all cells, calcein-am is used as an intensely green fluorescent indicator of viable cells, and propidium iodide is used as a red fluorescent indicator of cells with compromised membranes. A. SH-SY5Y cells treated with 1.6-µM ionomycin B. SH-SY5Y cells treated with 25-µM ionomycin Fig 1. Human neuroblastoma cells (SH-SY5Y) treated with ionomycin (A. 1.6 µm; B. 25 µm) for 4 h in complete culture medium, prior to reagent addition. Images acquired on the IN Cell Analyzer 3000 represent 40% actual area of a field of view. The nuclei of all cells exhibit blue fluorescence due to Hoechst 33342. The nuclei of cells with compromised membranes (undergoing cytotoxicity) exhibit red fluorescence due to propidium iodide. Viable cells exhibit green fluorescence due to the conversion of calcein-am to calcein by intracellular esterases. Biological protocol and image acquisition Cells in log-phase growth were seeded into 96-well microplates (5000 cells/well) and incubated in culture medium for 24 h at 37 C, 5% CO 2. Culture medium was removed from the cells and replaced with culture medium (100 µl) containing test compounds at appropriate concentrations (n = 8). Cells were incubated for 1 h, 4 h, or 24 h at 37 ºC, 5% CO 2. A solution containing Hoechst 33342, propidium iodide, and calcein-am was prepared in culture medium (50 or 100 µl) and added directly to the contents of each well to provide final concentrations of 10-µM Hoechst 33342, 10-µM propidium iodide, and 50-nM calcein-am. For a two-color assay, either calcein-am or propidium iodide can be omitted. Note: The assessment of cell viability is a live-cell assay; removal of culture medium from wells or fixation of cells after treatment with test compounds should be avoided since dead and poorly attached cells will be detached by this process. After incubation of cells and reagents at 25 ºC for 10 min, images were acquired on the IN Cell Analyzer 3000: excitation was provided by 488-nm (calcein-am and propidium iodide) and 364-nm (Hoechst 33342) laser lines with a neutral density filter of 2 applied to both lasers; emission was monitored through 535/45-nm, 635/55-nm and 450/65-nm emission filters, respectively with a camera exposure time of 1.7 ms. Note: Most countries have legislation governing the handling, use, storage, disposal, and transportation of mammalian cell lines. Users must be aware of and observe the Local Regulations or Codes of Practice, which relate to such matters. Image analysis Images were analyzed directly using the IN Cell Analyzer 3000 Cell Viability Analysis Module (Fig 2). The Hoechst 33342 signal was used to produce an object mask and each cell was classified according to the relative fluorescence intensity of calcein-am and propidium iodide. Outputs from the algorithm include cell number, the percentage of live and dead cells, and key phenotypic characteristics of these sub-populations. Run file selection Selection of marker and signal sampling channels Object definition and object filtering Sampling area definition and signal filtering Automated analysis Selection and application of cell viability classification parameters Data output Fig 2. Analysis flow-chart for the Cell Viability Analysis Module showing key steps in analysis of images. 2 Application Note 28-4070-51 AA 2006-07
Results Use of the IN Cell Analyzer 3000 has enabled the development of a simple, homogeneous fluorescent cell viability assay format which can be applied to a range of cell lines to generate the effective cytotoxic dose for a number of compounds (Fig 3 and Table 1). In addition, to the measurements associated with viability (calcein-am) and cytotoxicity (propidium iodide), cell number also provided a good indication of cytotoxicity. values obtained using calcein-am, propidium iodide, and cell number showed good correlation after 4-h exposure to compounds (Fig 3). The time taken for full-field image acquisition and online analysis of the three-color assay for a 96-well microplate was 9 min, whilst re-analysis of images required 48 s per plate. Table 1. Effective cytotoxic concentrations of four compounds on four cell lines viability and cytotoxicity (µm)* Cell line Ionomycin Staurosporin Vinblastine Paclitaxel HeLa 143 > 100 474 282 A549 20 Not done 460 Not done U-2 OS 5 > 100 488 249 SH-SY5Y 19 Not done 212 > 500 * Cell lines were exposed to compounds for 4 h in complete medium (n = 8) prior to image acquisition and analysis on the IN Cell Analyzer 3000. values were derived for the viability (% live) and cytotoxicity (% dead) data (Fig 3). Values are outside assessed concentration range. The use of complete media in cell viability assays provided a more relevant cellular environment and permitted prolonged compound exposure times than would be feasible in buffer-based systems (Fig 4). In addition, cellular imaging allowed the measurement of simple phenotypic characteristics of cells that would be unavailable using flow cytometer or plate readerbased assays. values for ionomycin derived from cell number and viability curves after 4-h and 24-h exposure were equivalent, indicating ionomycin causes a cytotoxic event resulting in rapid cell death. However, prolonged compound exposure and the provision of phenotypic data can facilitate the differentiation of cytotoxic and cytostatic effects and assist characterization of subtle cytotoxic agents (Fig 4 and Table 2). For example, after 4-h and 24-h exposure to relatively high concentrations (> 100 µm) of paclitaxel, the cytotoxic effects of paclitaxel resulted in a decrease in cell viability (% live cells) and average object area (Fig 4). However, exposure of cells to lower concentrations of paclitaxel (10 100 nm) for 24 h resulted in a decrease in cell number but a reciprocal increase in object area. Cell number has decreased without measurable cytotoxicity through the lower concentration range due to the cytostatic effect of paclitaxel; the induction of cell cycle arrest at G2/M phase has resulted in a population of cells with increased genomic complement (predominantly 4n) and a consequent increase in average nuclear area. Fig 3. Cell viability assays employing Hoechst 33342, calcein-am, and propidium ioidide and analyzed using the Cell Viability Analysis Module. SH-SY5Y, HeLa, and U-2 OS cells were treated with ionomycin, vinblastine, or paclitaxel, respectively for 4 h in complete culture medium, prior to reagent addition. Images were acquired on the IN Cell Analyzer 3000. Black, green, and red symbols denote cell number, % live cells, and % dead cells, respectively (mean ± SE for 8 repeats). values of 19 µm, 474 µm and 249 µm, respectively were obtained for each of the compounds using the viability and cytotoxicity curves (% live and % dead). values of 20 µm, 480 µm, and 163 µm, respectively were obtained for each of the compounds using the cell number curves. 3 Application Note 28-4070-51 AA 2006-07
Fig 4. Cell viability assays analyzed using the Cell Viability Analysis Module. SH-SY5Y cells were treated with ionomycin or paclitaxel for 4 h or 24 h in complete culture medium, prior to reagent addition. Images were acquired on the IN Cell Analyzer 3000. Black, green, and red symbols denote cell number, % live cells, and % dead cells, respectively (mean ± SE for 8 repeats). Object area data (blue symbols) has been included on the paclitaxel graphs. values are presented in Tables 1 and 2. Table 2. Effective cytostatic and cytotoxic concentrations of four compounds on four cell lines. Ionomycin Staurosporin Vinblastine Paclitaxel Cell line* Cell number Viability Cell number Viability Cell number Viability Cell number Viability HeLa 14.5 26.7 0.459 7.06 < 1.50 136 < 0.750 316 A549 10.3 19.6 Not done ND < 0.010 154 Not done ND U-2 OS 14.6 31.7 Not done ND < 1.50 195 < 0.750 318 SH-SY5Y 5.8 13.8 Not done ND < 0.010 125 0.029 184 * Cell lines were exposed to compounds for 24 h in complete medium (n = 8) prior to image acquisition and analysis on the IN Cell Analyzer 3000. values for cell number data (representing cytostatic and cytotoxic effects) and viability data (% live and % dead cell data; representing cytotoxic effects only) are shown in fig 4. Values are outside assessed concentration range. Note: The assay format, method, and dye concentrations have been optimized for Hoechst 33342, calcein AM, and propidium iodide. Dying and dead cells may demonstrate low levels of fluorescence with nucleic acid fluorophores due to DNA fragmentation and nuclease activity during apoptosis and necrosis. However, the use of ethidium homodimer as an alternative to propidium iodide resulted in an additional reduction in the Hoechst 33342 signal precluding accurate object definition and identification of dead cells (data not shown). Ethidium homodimer is a bisintecalating agent and in cells with compromised membranes, it may compete for, or prevent access of Hoechst 33342 to DNA binding sites, or directly quench the signal to a much greater extent than propidium iodide. Application Note 28-4070-51 AA 2006-07 4
Conclusion The acquisition of images from cell viability assays using the IN Cell Analyzer 3000 provides a simple, rapid, homogeneous, and highly informative fluorescent cell viability assay. The sequential process of exposure of cells to compounds and reagents, and immediate live-cell image and data acquisition can be transcribed into suitable automated and integrated workflow systems with standard liquid-handling dispensing units. The flexibility of the system has been demonstrated with a number of cell lines, incubation times and compounds, and the speed and information content provided indicates that such assays may be a valuable initial step in high-content cellular screening strategies. Application Note 28-4070-51 AA 2006-07 5
Asia Pacific Tel: +85 65 62751830 Fax: +85 65 62751829 Australasia Tel: +61 2 8820 8299 Fax: +61 2 8820 8200 Austria Tel: 01 /57606 1613 Fax: 01 /57606 1614 Belgium Tel: 0800 73 890 Fax: 02 416 8206 Canada Tel: 1 800 463 5800 Fax: 1 800 567 1008 Central & East Europe Tel: +43 1 972 720 Fax: +43 1 972 722 750 Denmark Tel: +45 70 25 24 50 Fax: +45 45 16 2424 Eire Tel: 1 800 709992 Fax: +44 1494 542010 Finland & Baltics Tel: +358 9 512 3940 Fax: +358 9 512 39439 France Tel: 01 69 35 67 00 Fax: 01 69 41 98 77 Germany Tel: 0800 9080 711 Fax: 0800 9080 712 Greater China Tel: +852 2100 6300 Fax: +852 2100 6338 Italy Tel: 02 26001 320 Fax: 02 26001 399 Japan Tel: 81 3 5331 9336 Fax: 81 3 5331 9370 Korea Tel: 82 2 6201 3700 Fax: 82 2 6201 3803 Latin America Tel: +55 11 3933 7300 Fax: +55 11 3933 7304 Middle East & Africa Tel: +30 210 96 00 687 Fax: +30 210 96 00 693 Netherlands Tel: 0800-82 82 82 1 Fax: 0800-82 82 82 4 Norway Tel: +47 815 65 777 Fax: +47 815 65 666 Portugal Tel: 21 417 7035 Fax: 21 417 3184 Russia, CIS & NIS Tel: +7 495 956 5177 Fax: +7 495 956 5176 Spain Tel: 902 11 72 65 Fax: 935 94 49 65 Sweden Tel: 018 612 1900 Fax: 018 612 1910 Switzerland Tel: 0848 8028 10 Fax: 0848 8028 11 UK Tel: 0800 515 313 Fax: 0800 616 927 USA Tel: +1 800 526 3593 Fax: +1 877 295 8102 www.gehealthcare.com/lifesciences GE Healthcare Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK General Electric Company reserves the right, subject to any regulatory approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Representative for the most current information. 2006 General Electric Company - All rights reserved. GE and GE Monogram are trademarks of General Electric Company. Amersham, and Amersham Biosciences are trademarks of GE Healthcare Limited. Hoechst is a trademark of Hoechst AG; Viewplate is a trademark of Perkin Elmer, Inc. The IN Cell Analyzer 3000 is the subject of US patents 6 400 487 and 6 388 788 and US patent application number 10/227552, together with other granted and pending family members, in the name of GE Healthcare Biosciences Corporation. The IN Cell Analyzer 3000 and associated analysis modules are sold under license from Cellomics Inc. under US patent Nos 6573039, 5989835, 6671624, 6416959, 6727071, 6716588, 6620591, 6759206; Canadian patent No 2328194, 2362117, 2,282,658; Australian patent No 730100; European patent 1155304 and other pending and foreign patent applications. imagination at work Application Note 28-4070-51 AA 2006-07