CytoTox 96® Non-Radioactive Cytotoxicity Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
If the target cells are not completely lysed (as determined by microscopy), add another 5µl of Lysis Solution (10X).	Add 50µl of CytoTox 96® Reagent to each well and cover the plate with foil or an opaque box to protect it from light and incubate for 30 minutes at room temperature	If low % cytotoxicity is observed, increase the incubation time with the cytotoxic cells from 4 hours to 6–8 hours

Upstream tips
If the target cells are not completely lysed (as determined by microscopy), add another 5µl of Lysis Solution (10X).
Protocol tips
Add 50µl of CytoTox 96® Reagent to each well and cover the plate with foil or an opaque box to protect it from light and incubate for 30 minutes at room temperature
Downstream tips
If low % cytotoxicity is observed, increase the incubation time with the cytotoxic cells from 4 hours to 6–8 hours

Publication protocol

The density of the cells leaking from the matrix, i.e., present in the reservoir, the pH, pCO2, pO2, osmolality, concentrations of glucose, lactate, glutamine, glutamate and ammonia, were measured by Bioprofile FLEX (Nova Biomedical). The perfusion rate was calculated based on the matrix volume (150 cm3) by weighting the feed medium and harvest bottles. The cell viability was determined by measuring the activity of lactate dehydrogenase (LDH) (cytotoxicity enzymatic assay, Promega) to determine the concentration of dead cells, Cdead, in the daily harvest samples. A standard curve of released LDH activity was made from lysing known numbers of cells. The viability was then calculated as
(2)
where Cv is the viable cell density measured by the biomass sensor, volmatrix is the matrix volume, volreservoir is the reservoir volume, D is the perfusion rate. The IgG quantification was done by high-performance liquid chromatography (HPLC) Protein A method (Protein A column, Applied Biosystems, USA). Daily samples were purified and concentrated with NAb Protein A/G spin kit (Thermo Scientific, USA) and reducing sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) was performed. The cell specific consumption/production rates of nutrients/metabolites, the accumulated IgG production, the cell specific productivity and the volumetric productivity were calculated as previously described (Clincke et al., 2013a, Clincke et al., 2013b).

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Manufacturer protocol

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

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