Validation of cell density and viability assays using Cedex automated cell counter (original) (raw)
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Background: In this study we developed a method to measure cell concentration and viability in specimens received in flow cytometry and cytogenetics laboratories. Methods: Specimens are stained with a vital fluorescent dye, SYTO13, the cell impermeant viability dye, 7-AAD, and a leukocyte marker, CD45. After the addition of an internal calibrator microsphere, FLOW-COUNT TM , the flow cytometer is capable of measuring the viability of nucleated cells, giving a general assessment of leukocyte populations and measuring their concentration. Results: An accurate assessment of specimen quality is an important parameter when performing flow cytometric and cytogenetic leukemia/lymphoma assessment. High quality specimen is desired to avoid the pitfalls of non-specific staining and limited cellularity/viability. Conclusions: Use of a cell count and viability measurement prior to leukemia and lymphoma assessment by flow cytometry and cytogenetics helps to increase the rate of successful immunophenotypic and cytogenetic analysis.
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This chapter is an introductory overview of the most commonly used assay methods to estimate the number of viable cells in multi-well plates. This chapter describes assays where data are recorded using a plate-reader; it does not cover assay methods designed for flow cytometry or high content imaging. The assay methods covered include the use of different classes of colorimetric tetrazolium reagents, resazurin reduction and protease substrates generating a fluorescent signal, the luminogenic ATP assay, and a novel real-time assay to monitor live cells for days in culture. The assays described are based on measurement of a marker activity associated with viable cell number. These assays are used for measuring the results of cell proliferation, testing for cytotoxic effects of compounds, and for multiplexing as an internal control to determine viable cell number during other cell-based assays.
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Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed 1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate 2. To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry 1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments 1. The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection 3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cellsensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.
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Journal of the Association for Laboratory Automation, 2007
T his review assesses the quality of the data acquired over a 13-week period from a High-Content Analysis screening project that used 297 unique cell lines. This article also evaluates the proficiency of a ''tipless'' (i.e., does not use disposable tips) full-automation design used for this project that prioritizes intralab system mobility and system configuration mutability. The request to assay a large number of cell lines with poorly characterized growth rates led us to devise an MDS PharmaServices, Inc. proprietary algorithm in an effort to select the proper cell plating density for each cell line. The performance metrics include coefficients of variation (CVs) of Controls for the cell plating data and Data Set Mean CVs for assessing replicate propinquity (i.e., how close the replicates are to each other). The performance of the automation system and our algorithm for this project produced data of superior quality. ( JALA 2007;12:318-26)
A simple method to measure cell viability in proliferation and cytotoxicity assays
Brazilian Oral Research, 2009
Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H + to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm) and green (500 to 600 ηm) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r 2 = 0.996; p < 0.01) and with the cellular concentrations (r 2 = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.