The ER Stress Monitoring Kit uses the naturally secreted Gaussia luciferase (Gluc) as a reporter. Other uses of the Gluc reporter include: monitoring of protein processing through the secretory pathway; cell viability, growth, proliferation and death. This reporter has several advantages over existing reporters used to monitor ER-stress, such as the secreted alkaline phosphatase (SEAP) and metridia luciferase. The Gaussia luciferase is 20,000-fold more sensitive than the SEAP assay with a linear range covering over 5 orders of magnitude with respect to cell number. Gluc uses coelenterazine as a substrate and its measurement is performed within seconds by simply taking a few microliters of conditioned medium, adding coelenterazine and acquiring a photon count using a luminometer (for 10 sec). On the other hand, the SEAP assay requires sample dilution, processing and incubation time which takes around 1.5 to 2 hrs to get the results. Also, the linearity of the SEAP assay with respect to cell number covers only 3 orders of magnitude after which the signal becomes constant. This could lead to false-negative results when the number of cells is above the linear range. Our laboratory uses the Gluc reporter very often and has also compared it to metridia luciferase. In our hands, metridia luciferase seems to be toxic to mammalian cells upon transfection. In addition, the Gluc is still over 300-fold more sensitive than metridia luciferase.
Several versions of this kit are available. One kit contains the plasmid DNA expressing Gluc and its substrate coelenterazine. This kit requires transfection reagents to deliver the plasmid DNA into mammalian cells. Another kit contains lentivirus vector expressing Gluc along with its substrate coelenterazine. An additional advantage of this kit is that lentivirus vectors are able to transduce many cell types very easily by simply incubating the cells with some of the virus as well as polybrene for few hours. The advantage of this system is that once cells are transduced with this vector, they will stably express the reporter and, therefore, one can freeze these cells and use them continuously without the need of cell transfection before each experiment. After gene delivery, cells are plated in triplicates in wells of either 24, 48 or 96-well plates. Twenty-four hours later, cells are washed once with PBS and treated with an ER-stress inducer such as dithiothreitol (DTT). At different time points, 10 or 20 ul aliquots of the conditioned medium are then transferred into a clean 96-well plate into which 50 ul 20 uM coelenterazine is added and light output is measured using a luminometer.
The Gluc assay to monitor ER-stress is highly sensitive, reproducible, cost-effective and easy to use. All it requires is its substrate, coelenterazine, and a luminometer.