Which common methods are used to measure cytotoxicity?
There are two commonly used methods of estimating dead cells; both methods take advantage of the loss of membrane integrity and the ability of indicator molecules to partition into a compartment not achievable if the cell membrane is intact. As illustrated in the following image, assays used to detect dead cells include measuring the leakage of a component (usually an enzyme marker) from the cytoplasm into the culture medium or the penetration of an otherwise non-permeable dye into cells with a compromised membrane.
Promega products: CytoTox-Glo™ Cytotoxicity Assay (Cat.# G9290), CytoTox-Fluor™ Cytotoxicity Assay (Cat.# G9260)
How does trypan blue staining work?
The selective staining of dead cells with trypan blue and microscopic examination on a hemocytometer is one of the most frequently used routine methods to determine the cell number and percent viability in a population of cells. The general concept is that trypan blue is excluded from live cells but penetrates dead cells with a damaged plasma membrane.
The trypan blue staining technique is usually performed on a single sample or relatively small numbers of samples from simple experiments. The main disadvantages of this technique are: the error involved with measuring a single sample, the subjective judgement of the user to determine what is a dead cell or stained debris, inconsistency among operators, and the time and manual labor involved with measuring multiple samples.
How do I choose a fluorescent DNA-binding dye for my cytotoxicity assay?
Fluorescent DNA binding dyes are generally nonpermeable to viable cells and can be used to detect the accumulation of dead cells in culture using a multiwell plate format. The most important and practical factors to consider when choosing a dye include: the emission wavelength, selectivity for staining DNA, cell permeability, solubility at the vendor-recommended concentration, detection sensitivity and cytotoxicity. Fluorogenic DNA dyes that readily pass through the intact cell membrane and stain the nucleus of live cells should not be used for measuring dead cells.
Promega product: CellTox™ Green Cytotoxicity Assay (Cat.# G8741)
Can I measure cytotoxicity in real-time?
Yes, DNA-binding dyes can be used to measure dead cells over time, However, it is important to consider whether prolonged exposure of the cells to the dye will affect their health or responsiveness. The figure below shows the effects of three different DNA-binding dyes continuously exposed to four different cell types for 72 hours before measuring cell viability using an ATP assay.
How do lactate dehydrogenase (LDH) assays work?
The presence of dead cells that have lost membrane integrity can be detected by measuring markers that leak from the cytoplasm into the culture medium. The most common marker used for this type of assay is lactate dehydrogenase. Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate and in the process, converts NAD+ to NADH. The reducing capacity of NADH can be used to reduce a variety of substrate molecules into products that are either colored, fluorescent, or luminogenic. Figure 5 illustrates the general scheme and assay chemistry used to detect LDH-release from the cytoplasm of dead cells. An excess amount of lactate and NAD+ as substrates are delivered in a reagent mixture to drive LDH to generate pyruvate and NADH. The reducing power of NADH is used to convert the substrate (resazurin) into the fluorogenic product (resorufin).
Colorimetric versions of this assay chemistry have used a tetrazolium compound as the diaphorase substrate which is converted into an intensely colored formazan product that can be measured using a spectrophotometer. Similarly, a luminometric assay can use a “pro-luciferin” substrate which is converted into a luciferin product that is linked to a firefly luciferase reaction to generate a luminescent signal. The colorimetric version of the assay was developed decades ago and lacks detection sensitivity. In addition, because of buffer incompatibility with live cells, it requires removal of culture supernatant to a different container to perform the assay. The fluorescent assay protocol is homogeneous and more sensitive than the colorimetric version. The luminogenic version of the assay is far more sensitive than the fluorogenic version, and enables sampling of 2–5 µl of culture supernatant at various times which can be stored frozen for future analysis of LDH release over time.
Promega products: LDH-Glo™ Cytotoxicity Assay (Cat.# J2380), CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Cat.# G7890)
Download the Assay Guidance Manual to learn more about cytotoxicity assays.