Measuring necrosis in eukaryotic cells is a common procedure and can be performed in a variety of ways. We work with bacterial pathogens known to have deleterious effects on mammalian cells and we typically study cell death of various cell lines including HeLa, Caco-2, Cos-7 and J774 macrophages. To study necrosis, we use a combination of both visual examination (i.e. trypan blue staining) and biochemical analysis, such as the activity of the cytoplasmic enzyme lactate dehydrogenase (LDH), which is released into the supernatant of necrotic cells following rupture of the plasma membrane. We initially chose the LDH Cytotoxicity Assay Kit from Biovision due to its low cost and because the protocol, which is on the company’s website, seemed easy to follow.
The LDH kit is stored at -20ºC and, providing it is left in the dark, is stable for several months. Before use, it requires the mixing of a catalyst with the dye reagent. We found it is a good idea to aliquot the dye solution into 5 mL fractions as it takes more than 45 min to thaw the main bottle to room temperature each time you use it. Also we noted that even a small amount of light exposure will alter the color of the dye, so ensure the aliquots are well light-protected.
Following experimentation, our adherent cell lines are tested for the release of LDH by sampling the extracellular medium above the cells with the kit. The great advantage of measuring cell death in this way is that it’s entirely non-invasive and cell death can therefore be monitored on the same samples over long time periods. The kit requires 100 µL of cell supernatant which is centrifuged to remove cellular debris and then transferred to a 96-well plate. This is then mixed with 100 µL reaction solution containing the LDH substrate, which turns red and can be measured at 500 nm. The enzyme reaction occurs at room temperature and takes about 30 min. We found that if the reaction is left too long, the absorbance will be much too high and cannot be read accurately, so it is important to keep monitoring the developing substrate. As a positive control (100% necrosis), we lyse all the cells with 0.5% triton and dilute this supernatant about 1:10 as the absorbance is always too strong.
We have been using this kit successfully for several years and are pleased with the results. We find the control cells (both positive and negative) always give very consistent readings. However, sometimes we find unusual or variable results with experimental cell samples and to date we do not know why this occurs. In particular, we have sometimes noted LDH activity going down over time rather than up suggesting possible degradation of this enzyme under certain conditions. LDH however, is known to be a very stable enzyme and is generally a good marker for necrosis so we are a little puzzled by this. We would recommend this kit to researchers wanting to measure necrosis in eukaryotic cells; the only item required (outside of the kit) is a spectrophotometer capable of reading near 500 nm. The non-invasive nature of the kit makes it a very simple and versatile way of studying necrosis and also permits additional analyses of the cells by other methods.