Continuous Analysis of ER Stress
Endoplasmic reticulum (ER) stress is associated with a variety of pathologies, including viral infections, ischemic injury, neurodegenerative diseases, and metabolic disorders. the detection, quantification, and monitoring of ER stress are necessary to study the molecular events involved in these pathologies.
However, conventional assays of ER stress do not allow continuous or sequential monitoring of ER stress in living cells and animals. Here, Creative Bioarray can detect and continuously monitor ER stress status in vitro and in vivo in real-time with the SEAP reporting system.
Technology Principles
Secretory alkaline phosphatase (SEAP), a mutant of human placental alkaline phosphatase, is best characterized by its ability to be secreted extracellularly by the expressing cell. The assay can be repeated and dynamic at any point in time by taking cell culture supernatant without destroying the cells, and the cells being assayed can continue to be used for other purposes. The sensitivity of the SEAP assay can be further improved by using a luminescence assay. This method allows for rapid, quantitative, sensitive, and continuous monitoring of ER stress in the same living cells. In addition, the method is useful for real-time monitoring of ER stress in animals in pathological situations.
In vitro ER Stress Real - Time Monitoring Service
Including but not limited to porcine kidney proximal tubular cell line, murine hepatocellular carcinoma cell line, rat alveolar macrophages
- Recombinant SEAP Preparation
SEAP-secretable cells were generated by stable transfection of cells. The expression of transgenes and transgene products was analyzed by northern blotting, and protein blotting.
- Chemiluminescence analysis of endoplasmic reticulum stress
The translated SEAP gene product is secreted from the cells into the medium, so it can be easily detected in the medium samples without disrupting the cells and the sample preparation is not time-consuming. Because lysis of cells is not required, sampling of cell culture media or other subsequent analyses can be repeated. SEAP reporter gene assay, chemiluminescence based on dioxetane CSPD.
The magnitude of SEAP reduction is proportional to the degree of ER stress and inversely proportional to the induction of the endogenous ER stress markers grp78 and grp94.
The technique allows for rapid detection of ER stress in vitro. SEAP activity can be sensitively and quantitatively detected using conventional chemiluminescence systems, and only 5 µl of culture medium or serum is sufficient for evaluation.
The response is very fast and exposure to triggers <45 minutes was found to be sufficient to detect ER stress in cultured cells.
In vivo ER Stress Real - Time Monitoring Service
In in vivo monitoring experiments, simple blood sampling without sacrificing the animal is sufficient to monitor ER stress. This means that the kinetics of ER stress can be easily monitored in the same animals during the disease process. Continuous in vivo monitoring of ER stress in specific tissues or organs by simple blood sampling would also be feasible if mice expressing SEAP under the control of tissue or cell type-specific promoters were generated. overexpression of SEAP by itself does not trigger ER stress in mammalian cells.
Service Applications
The service can be used to
- Help clients elucidate the relationship between ER stress and disease
- Screen drugs for therapeutic interventions in ER stress-related diseases
- In vivo studies requiring continuous monitoring of ER stress during the disease process
Creative Bioarray is dedicated to providing high-quality products, comprehensive services, and tailored solutions to support and facilitate life sciences and pharmaceutical research and development. If you have any questions or needs, please contact us, and our customer service staff will help you the first time.
All services and products are for scientific use only, not for medical use！
