CellTiter-Glo® Luminescent Cell Viability Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- cells were pretreated with Klotho for 4 h before the addition of different concentrations of glutamate or Aβ.	- After 24h of the pre treatment, cells were treated with CellTiter Glo reagent and incubated for 10min at RT.

Upstream tips
- cells were pretreated with Klotho for 4 h before the addition of different concentrations of glutamate or Aβ.
Protocol tips
- After 24h of the pre treatment, cells were treated with CellTiter Glo reagent and incubated for 10min at RT.

Publication protocol

For cell viability assays, cells were plated in 96-well plates in five repeats and allowed to attach for 24 h. The next day, the cells were pretreated with Klotho for 4 h before the addition of different concentrations of glutamate or Aβ. Twenty-four hours later, cell death was assessed using the CellTiter-Glo luminescent cell viability assay (Promega), which measures the production of ATP, according to the manufacturer's instructions. Briefly, 100 μl of CellTiter Glo reagent was added to an equal volume of cell culture medium present in each well. The contents were mixed for 2 min, and after stabilization of the signal at room temperature for 10 min, the luminescence was recorded on a microplate reader (Glomax Multi Detection System, Promega). Cytotoxicity was calculated by the following formula, as described previously (38).

In addition, cell death was assessed directly using the CytoTox 96® non-radioactive cytotoxicity assay, Promega), which is a colorimetric alternative to the 51Cr release cytotoxicity assays. The CytoTox 96® assay quantitatively measures lactate dehydrogenase, a stable cytosolic enzyme that is released upon cell lysis. The intensity of the red color formed in the assay was measured at a wavelength of 490 nm and was proportional to lactate dehydrogenase activity and to the number of damaged cells. The data were normalized to the activity of lactate dehydrogenase released from glutamate-treated culture medium (100%) and expressed as a percentage of this control.

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Manufacturer protocol

Download the product protocol from Promega for CellTiter-Glo® Luminescent Cell Viability Assay below.

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