Cytotoxicity Assays: Determining the Number of Live and Dead Cells in a Cell Population, MultiTox-Fluor Multiplex Cytotoxicity Assay:
Cell-based assays are important tools for contemporary biology and drug discovery because of their predictive potential for in vivoapplications. However, the same cellular complexity that allows the study of regulatory elements, signaling cascades or test compound bio-kinetic profiles also can complicate data interpretation by inherent biological variation. Therefore, researchers often need to normalize assay responses to cell viability after experimental manipulation. Although assays for determining cell viability and cytotoxicity that are based on ATP, reduction potential and LDH release are useful and cost-effective methods, they have limits in the types of multiplexed assays that can be performed along with them. The MultiTox-Fluor Multiplex Cytotoxicity Assay (Cat. No. G9200, G9201, G9202) is a homogeneous, single-reagent-addition format that allows the measurement of the relative number of live and dead cells in a cell population. This assay gives ratiometric, inversely proportional values of viability and cytotoxicity that are useful for normalizing data to cell number. Also, this reagent is compatible with additional fluorescent and luminescent chemistries.
A variety of methods are available for detecting apoptosis to determine the mechanism of cell death. The Caspase-Glo® Assays are highly sensitive, luminescent assays with a simple “add, mix, measure” protocol that can be used to detect caspase-8, caspase-9 and caspase-3/7 activities. If you prefer a fluorescent assay, the Apo-ONE® Homogeneous Caspase-3/7 Assay is useful and, like the Caspase-Glo®Assays, can be multiplexed with other assays. A later marker of apoptosis is TUNEL analysis to identify the presence of oligonucleosomal DNA fragments in cells. The DeadEnd™ Fluorometric and the DeadEnd™ Colorimetric TUNEL Assays allow users to end-label the DNA fragments to detect apoptosis
A cell counter is essential for quantitative growth kinetics, and a great advantage when more than two or three cell lines are cultured in the laboratory. The Countess; Automated Cell Counter is a bench-top instrument designed to measure cell count and viability (live, dead, and total cells) accurately and precisely in less than a minute per sample, using the standard Trypan Blue uptake technique. Using the same amount of sample that you currently use with the hemacytometer, the countess. Automated Cell Counter takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells.
The latest generation of cell-based assays includes luminescent and fluorescent chemistries to measure markers of cell viability, cytotoxicity and apoptosis, as well as to perform reporter analysis. Using these tools researchers can investigate how cells respond to growth factors, cytokines, hormones, mitogens, radiation, effectors, compound libraries and other signaling molecules. However, researchers often need more than one type of data from a sample, so the ability to multiplex, or analyze more than one parameter from a single sample, is desirable.
Hemacytometers may be obtained from most major laboratory suppliers (e.g., Baxter Scientific). The procedure below provides some general directions on how to use the hemacytometer.
1. Clean the chamber and cover slip with alcohol. Dry and fix the coverslip in position.
2. Harvest the cells. Add 10 μL of the cells to the hemacytometer.
Do not overfill.
3. Place the chamber in the inverted microscope under a 10X objective. Use phase contrast to distinguish the cells.
4. Count the cells in the large, central gridded square (1 mm2). The gridded square is circled in the graphic below. Multiply by 104 to estimate the number of cells per mL. Prepare duplicate samples and average the count.