New luminescence-based approach to measurement of luciferase gene expression reporter activity and adenosine triphosphate-based determination of cell viability.
The assay employing firefly luciferase as the end-point reporter is one of the most popular gene reporter systems. However, the physiological conditions of cells may affect the reporter gene expression, which makes an assessment of cell viability desirable. Estimates of cell viability may be based on different principles. We tested for correlations between various cell viability assessments, including luminescent determination of adenosine triphosphate in whole-cell lysate, and the reporter luciferase activity in pluripotent embryonic and colon adenocarcinoma cells. Luciferase activity in cell lysate from both cell lines cultured under different conditions correlated with the amount of viable cells assessed by all of the methods employed. Importantly, it was also possible to carry out adenosine triphosphate determination in cell lysates prepared in the buffer originally designed for determining luciferase activity; it correlated significantly with adenosine triphosphate determination in cells lysed in the buffer originally designed for adenosine triphosphate determination. The results suggest that the assessment of live cells by determining adenosine triphosphate can be multiplexed with a luciferase reporter gene assay, which allows independent monitoring of both reporter expression and cell viability.
Konopka
R
R
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
Hýzdalová
M
M
Kubala
L
L
Pacherník
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D045744:Cell Line, Tumor; D002470:Cell Survival; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*
D000255:Adenosine Triphosphate; D008156:Luciferases