Since LDH assay determines membrane integrity, may this test be sufficient to conclude that there is a necrosis cell death-related induced by drugs?
I second Efrain's comment. Nobody will question the LDH level is relating to cell death (bot necrosis and apoptosis) but to say it is due to necrosis will be a concern.
It would be best to try and modulate the amount of cell death and try to home in on a mechanism. For example, caspase inhibitors or necroptosis inhibitors such as necrostatin can help determine the difference between various pathways. If no canonical inhibitory treatments help reduce the amount of LDH then you might conclude actual, accidental necrosis is happening.
No sangue ou em células isoladas?
Se fizer um ensaio com células, vc pode sugerir que está ocorrendo necrose; porém vc não pode diferenciar esta necrose de outro tipo de morte celular...
Jah se vc aferiu no sangue pior ainda, pois vc não consegue definir em qual tecido pode estar havendo morte celular ou não...
I repeatedly had to make the experience, that relying on a singular marker of cell death (whether being apoptotic or necrotic) may be critical and lead to false conclusions: For cell death in general (as a combined measure of apoptosis and necrosis) LDH may be an excellent parameter to start with. I would recommend to add a marker reflecting metabolic turnover like MTT, XTT or alamar blue. You will be surprised about the low (inverse) correlation between MTT and LDH is in some cases. To differentiate necrosis from apoptosis, during my active phase in the lab I usually employed caspase-3 assays, which can be nicely multiplexed with other markers (and the right equipment), e.g. in microscopy. Annexin is certainly also a good choice. The cell count procedures can be largely automated. Using a microscope offers the additional advantage, that you can visually evaluate the typical morphologic signs of apoptosis or even observe ongoing apoptosis in real-time (e.g. with fluorogenic Caspase3/7 substrates.
Thanks to everyone who helped with the answers! This discussion has been very important to me.
Dear Luis Felipe,
As mentioned above, the LHD assay reflects disruption of plasma membrane. It may happen during necrosis but also it reflects late apoptosis. If you want to distinguish between these cell death modes you have to utilise additional measurements. In addition to Annexin V staining (which is sometimes problemattic), I would recomend you to determine caspase-3 activation assay during the process to search for biochemical apoptotic hallmark and to examine the nuclear morphology for morphological hallmarks of apoptosis.
Good luck,
Petr Mlejnek,
Associate Professor in Biology
This test is not sufficient to conclude that there is a necrotic or necrotic like cell death induced by the used drugs. It just indicates that these cells are actually dead. You can conclude that this cell death was necrotic, provided that there were neither morphological (cell shrinkage, chromatin condensation, apoptotic bodies formation) nor biochemical signs of apoptosis (caspase-3 activation, endonuclease activation, externalization of phosphatydylserine). Instead, you should observe cell swelling, organele swelling, Ca2+ influx...
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