The Roche LDH Cytotoxicity Detection Kit is designed to be a simple method to directly quantify cell death in culture. The assay is based on the measurement of lactate dehydrogenase (LDH) released into the growth media when the integrity of the cell membrane is lost. My use of this kit was primarily with MG-63 human osteosarcoma cells, predominantly in assays aimed at determining cell death following drug dosing or the extent of cell death as cells differentiated in long term cultures. Usually, this assay was conducted in parallel to experiments aimed at assessing changes to other phenotypes in cells grown in the same manner.
To perform the assay, media is harvested from cultures and centrifuged to remove cell debris. Media can then be stored frozen (or at 4ºC for shorter periods of time), or the assay conducted immediately by mixing the media with the assay reagent which is prepared by mixing two separate solutions. This is incubated for 30 minutes, protected from light, and the absorbance is then read at 490 nm, with a reference reading at a wavelength above 600 nm.
As the assay is conducted on harvested media, it is extremely adaptable to a variety of culture conditions, methods and well sizes. In addition, separate phenotypic assays can be conducted on the discarded cell layer. In my case, I used the cell layer to measure cell number (cell counts, DNA content, protein content, etc.). This also allowed cytotoxicity readings to be standardized to cell number in order to yield a more accurate reflection of cell death, which is especially important over longer assay periods when cytotoxic agents act early, preventing cell growth. The kit also recommends appropriate controls and methods to determine parameters such as percentage cytotoxicity of your cells without the need to determine cell numbers. If you choose to disregard these controls, a control which should always be used is a well to which 10% triton-X-100 media is added just prior to harvest. This serves as an excellent positive control (100% cell lysis) and if you do detect positive readings for your samples, it allows these readings to be placed in the appropriate context.
The major limitation of this assay is that serum and some other compounds have inherent LDH activity. The fetal calf serum I used had extremely high background readings. The assay is therefore limited to serum-free or low-serum conditions, limiting the assay culture period (depending on your cells’ tolerance to low serum) and reducing the scope of the assay as it can no longer allow determination of cell death caused under normal growth conditions (i.e. in 10% fetal calf serum). At a minimum, you should always first test the assay with an unused aliquot of the media you intend to use and compare the reading to that from media lacking supplements (e.g. straight DMEM).
As a result, I would only recommend this product for short term cytotoxicity assays (such as screening for drug-induced cytotoxicity) in serum-free or low serum conditions. If these happen to be the conditions under which you test your compounds or conditions of interest, then the LDH assay from Roche will be easily adaptable to you needs.
PhD Student
School of Biomedical Sciences
University of Queensland