One of the main events that occur after cell death is the loss of membrane integrity, which allows chemicals or proteins to freely enter or exit the cell. Taking advantage of this phenomenon, we are able to evaluate the cytotoxicity of a given compound either by determining the exact number of dead cells stained with cell-impermeant fluorescent dyes or indirectly, by measuring the activity of enzymes that leak into the extracellular medium after cell membrane damage.
Membrane integrity dyes are cell-impermeant and only able to enter cells with a compromised plasma membrane. We use the nucleic acid-binding dyes (eg. DRAQ7, PI, 7-AAD, EthD-1, SYTOX) which are nonfluorescent in aqueous media but exhibit increased fluorescence upon binding to double-stranded DNA (dsDNA) or RNA. They allow for the fast and accurate evaluation of cytotoxicity and for multiplex experiments with other fluorescent dyes.
Enzyme leakage-based cytotoxicity assays detect extracellular glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH) or adenylate kinase (AK) released from damaged cells. Released enzymes activity can be quantified by a coupled enzymatic reaction in which provided substrates (lactate and NAD+, NADP+ or ADP) are converted to NADH, NADPH or ATP which further can be measured using different assay chemistries. The generated color, fluorescent or luminescent signal is proportional to the amount of ezyme present in analysed sample. There is no need for cell lysis so repeated samples of supernatant can be taken from wells over time without disrupting the cells themselves. This allows for kinetic analysis of cell death and multiplexing with other tests.
The essential cell toxicity assays are summarised in the table below:
| LDH-Glo™ | CyQUANT™ LDH | CellTox™ | |
| Working principle | LDH released from cells with damaged membrane is used for coupled reactions resulting in luciferin generation which is then converted by luciferase with luminescent signal emission. | LDH released from cells with damaged membrane is used for coupled reactions resulting in reduction of tetrazolium salts to insoluble formazan detected by colorimetry. | CellTox™ dye enters only cells with damaged cellular membrane. It gains fluorescent properties after complex formation with DNA. |
| What is detected | LDH released from cells | LDH released from cells | Impaired membrane integrity (exposed DNA) |
| Signal type | Luminescence | Colorimetric | Fluorescence |
| Platform | Plate reader | Plate reader | Plate reader / fluorescence microscopy |
| Sensitivity | 4/4 | 2/4 | 3/4 |
| Throughput | Medium | Low | Medium |
| Is cell lysis required? | no | no | no |
| End-point / real time | Real-time | Real-time | Real-time |
| Multiplexing | yes | yes | yes |
| Model system | adherent and suspension cell cultures | ||
In addition to the above most commonly used assays we also offer alternative approaches to suit specific experimental needs, see more info.
Example output:
