CytoTox 96® Non-Radioactive Cytotoxicity Assay

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	- Supernantant was incubated for 10min after adding substrate

Protocol tips
- Supernantant was incubated for 10min after adding substrate

Publication protocol

THP-1 monocytes or Caco-2 cells were plated and treated with different concentrations of OmpU (5, 7, and 10 μg/ml) or buffer for 24 h. After 24 h, cells were subjected to an LDH release assay. It is based on the principle that upon permeabilization of plasma membrane, LDH, which is a cytosolic enzyme, is released in the culture supernatants. Upon the addition of substrates like tetrazolium salts, LDH present in the culture supernatant converts the salt into a colored product. Intensity of the color produced is proportional to the number of lysed cells and can be measured spectrophotometrically. The LDH release assay was performed using the CytoTox 96® non-radioactive cytotoxicity assay kit (Promega Corp., Madison, WI) as per the manufacturer's instructions. Briefly, cells were pelleted, and supernatant for each treated sample was collected separately. Substrate provided in the kit was added to the supernatant of different samples. Supernatant from cells treated with lysis solution provided in the kit served as a positive control. Supernatants were incubated with substrate for 10 min. Following incubation, stop solution was added, and absorbance was measured at 490 nm.

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Manufacturer protocol

Download the product protocol from Promega for CytoTox 96® Non-Radioactive Cytotoxicity Assay below.

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