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Integrating cell health analysis into everyday workflow: by making cell health analysis and analyzers simple and affordable, cell-based experiments can be made more consistent and reproducible, enabling more productive research.

The assessment of cell concentration in combination with viability is an important step in the characterization of cell health. Cell concentration and viability information can be used for monitoring proliferation rates, optimizing growth conditions and normalizing cell data for further studies, such as assessing the impacts of cytotoxic compounds. Current methods rely on multiple, sometimes complex, instrument platforms to provide these answers, reducing flexibility, limiting the ability to simply obtain comprehensive cell health information and adding increased costs to researchers. Other simpler methods provide inconsistent results because of their dependence upon single-uptake dyes, which do not effectively discriminate between the various states of cellular demise. Therefore, there is a crucial need for analytical methods that provide rapid, robust and reproducible count and viability data to enable the efficient, daily execution of cellular research.

This article describes the use of EMD Millipore's Muse Cell Analyzer to provide multidimensional cell health analysis. The simplified format enables researchers to obtain a comprehensive picture of cellular health. Using multiparametric fluorescent detection of individual cells via microcapillary flow technology, the system enables highly sensitive and rapid detection of cellular samples using minimal cell numbers. The article shows that the assay provides superior performance to conventional viability and count measurement by Trypan blue exclusion.

Key features of the assay include accurate and precise data; mix-and-read protocols that allow for rapid measurement and instantaneous results; a proprietary combination of two fluorescent dyes that discriminate viable from dead, nucleated cells; and validation on a variety of both suspension and adherent cell lines.

Muse Count & Viability Assay uses a proprietary mix of two DNA intercalating fluorescent dyes in a single reagent to provide information on total cell concentration and viability. One of the dyes is membrane permeant and will stain all cells with a nucleus, allowing for the distinction of cellular debris from cells without a nucleus. The second dye only stains cells whose membranes have been compromised. Dying and dead cells stain with both dyes, but dying cells have a lower fluorescence intensity than do dead cells. Stained samples are then analyzed on the system using a guided touchscreen interface. The Count & Viability Assay displays results in an easy-to-read results page with an optional plot display. The use of dual fluorescent probes that clearly identify all nucleated cells, live and dead, allows for greater sensitivity and accuracy compared to colorimetric methods.

[FIGURE 1 OMITTED]

The touchscreen interface is shown in Figure 1. Briefly, a user enters the Count & Viability Module and hits "Run Assay." The touchscreen prompts the user to load a sample and, through simple on-screen instructions, guides the user through the optimization and verification of settings. The user then enters sample-specific information and touches "Run Sample." The instrument displays the results screen with the calculated concentration values and provides the user the option to view the dotplot as well as adjust markers between samples (Figure 1, bottom).

Result parameters include information on the number of viable cells/mL, percent viability, total cells/mL, total viable cells in original sample, total cells in original sample, dilution factor (input value), original volume (input value), sample number and sample ID.

Counting accuracy

The counting accuracy and linearity of the Muse Cell Analyzer was verified by measuring its ability to provide counts on multiple dilutions of reference counting beads. Figure 2 compares expected bead concentrations to bead concentrations measured using the Muse Cell Analyzer at multiple concentrations in the range of 1.0 x [10.sup.4] to 1.0 x [10.sup.6] beads/mL. Each point in the figure represents the average of triplicate samplings, and error bars represent corresponding standard deviations. The slopes and correlation coefficients of linear regression fit curves were both close to one, demonstrating that excellent counting accuracy and linearity can be obtained using the analyzer for the concentration range tested for reference counting beads.

[FIGURE 2 OMITTED]

Cell concentrations were determined across several cell lines, including both suspension and adherent lines, at a variety of concentrations. Figure 3 shows the comparison of observed vs. expected cell concentrations for five of the cell lines tested. The theoretical concentrations were calculated based on the serial dilution of the original cell sample, whose concentration was established using the Muse Cell Analyzer. The slopes and R2 values for all the cell lines tested closely approached one, demonstrating that the assay can provide linear responses across a wide range of cell concentrations, as well as diverse cell types.

Comparison

The accuracy of the Muse Count & Viability Assay was compared with other methods that provide count and viability information, including traditional methods that use Trypan blue staining, such as manual hemocy-tometer counts, as well as automated image-based analysis of Trypan blue-stained samples.

Five different cell lines at multiple concentrations and viabilities were analyzed using the Muse Count & Viability protocol and manufacturer-recommended protocols for each of the other methods. Researchers calculated the comparison of the average of triplicate measurements for each individual cell counting method versus the average cell concentration calculated by taking the mean average cell concentration from all three methods together. Regression statistics showed that the Muse Cell Analyzer demonstrates excellent agreement and provides accurate and comparable results to a variety of viability methods and instruments.

Precision and reproducibility

The precision of the Muse Count & Viability Assay was evaluated using the analysis methods and studies described earlier. Table 1 summarizes the average percent coefficient of variation (%CV) and %CV range obtained using the three methods to analyze 90 cellular samples from suspension and adherent cell lines at multiple concentrations.

The table demonstrates that the Muse Cell Analyzer provided average %CV of 4.0% for cellular concentration determination, which was lower than that observed for image-based automated counting (average %CV of 9.2%) and lower than that observed for manual hemocytometry (average %CV of 6.3%). While imagebased automated counting methods and manual hemocytometry displayed broader ranges of %CVs, the Muse Cell Analyzer exhibited a narrow range of %CVs and consistently provided %CVs less than 10% over the entire range of samples tested. Higher %CVs were observed for the Trypan blue-based methods, particularly at lower cell concentrations. The data demonstrated that the Muse Cell Analyzer can provide superior precision for cell counting measurements for multiple cell lines across multiple concentrations.

Table 1 also demonstrates that the Muse Cell Analyzer has a lower average %CV (2.2%) for viability measurements compared to the other methods. The %CV for viability measurements on the Muse Cell Analyzer was <7% for all samples.

[FIGURE 3 OMITTED]
               Cell                     Viability
             Conentration
Analysis     Average %CV   %CV Range  Average %  % CV Range
Method

Muse' Cell           4.0%   0.3-8.8%       2.2%    0.4-5.6%
Analyzer

Image-based          9.2%  1.2-23.3%       3.7%   0.8-12.1%
Automated
Counter

Manual               6.3%  0.5-15.3%       4.5%    0.5-9.2%
Hemocytometer

Figure (above): Comparison of observed uersus cell concentration
results for serial dilutions of five representative cell lines.
Table 1 (below): Data are based on triplicate measurements of 30
cellular sample from suspension and adherent cell lines at
multiple concentrations and viabilities.


Better, faster research

The Muse Cell Analyzer is a multifaceted instrument that enables measurement of multiple cell health-related parameters on a single platform. Specific assay modules facilitate rapid, easy assessment of cell health using assays for counting and viability, apoptosis detection and cell cycle distribution.

Performance data demonstrate excellent correlations with traditional, accepted analysis methods and confirm that this platform yields accurate results for a variety of cell types and concentrations. Furthermore, the Muse platform yields superior precision compared to traditional methods of cell counting and viability measurement. By making cell health analysis simple, affordable and easily accessible, the Muse Cell Analyzer can help integrate cell health analysis into everyday cell culture workflows. As a result, cell-based experiments can be made more consistent and reproducible, enabling faster, more accurate decisions for more productive research.

CONTACT

For more information, please visit EMD Millipore at www.millipore.com, or call 510-576-1400.

AT A GLANCE

* There is a crucial need for analytical methods that provide reproducible count and viability data for cellular research.

* Muse Count & Viability Assay uses a mix of two DNA intercalating fluorescent dyes.

* The Muse Cell Analyzer has a lower average %CV (2.2%) for viability measurements as compared to other methods.

by Katherine Gillis, Julie Clor, Asima Khan and Kamala Tyagarajan, HAD Millipore, Hayward, Calif
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Title Annotation:Feature
Author:Gillis, Katherine; Clor, Julie; Khan, Asima; Tyagarajan, Kamala
Publication:Laboratory Equipment
Date:Apr 1, 2012
Words:1390
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