Oxidative Stress Markers in Cell Culture

As a marker of lipid peroxidation, MDA was measured from the cell culture media with a specific colorimetric test. According to the protocol, 300 μL freshly prepared thiobarbituric acid (TBA) stock solution was mixed with 100 μL cell culture media. Solutions were incubated at 95 °C for 1 h, followed by 10 min cooling on ice. The absorbance of the samples was read at 532 nm with a Multiscan GO 3.2 reader.
Protein damage caused by oxidative stress was examined with a Protein Carbonyl ELISA Kit (Cat. No. MBS2600294, MyBioSource, San Diego, CA, USA) by measuring the protein carbonyl content of the cell lysate. To monitor the oxidative DNA damage, 8-OHdG concentration was assayed from the cell lysate with a specific 8-OHdG ELISA kit (Cat. No. MBS808265, MyBioSource, San Diego, CA, USA). The measurements were carried out according to instructions of the manufacturer’s protocol. The absorbances of the samples were read at 450 nm by a Multiscan GO 3.2 reader.
As a marker of endoplasmic reticulum stress, a chaperone protein—GRP78—was measured from cell lysate with a feline-specific ELISA kit (Cat. No. MBS072358, MyBioSource, San Diego, CA, USA), based on the instructions of the manufacturer’s protocol. The absorbance was read at 450 nm via a Multiscan GO 3.2 reader.