A novel quantitative flow cytometry-based assay for autophagy
العنوان: | A novel quantitative flow cytometry-based assay for autophagy |
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المؤلفون: | Gerald M. McInerney, Kai Er Eng, Marc D. Panas, Gunilla B. Karlsson Hedestam |
المصدر: | Autophagy. 6:634-641 |
بيانات النشر: | Informa UK Limited, 2010. |
سنة النشر: | 2010 |
مصطلحات موضوعية: | Autophagosome, Green Fluorescent Proteins, Intracellular Space, Endogeny, Nutrient sensing, Biology, Flow cytometry, Biological pathway, Mice, Cellular degradation, Autophagy, medicine, Animals, Humans, Molecular Biology, medicine.diagnostic_test, fungi, Chloroquine, Cell Biology, Saponins, Flow Cytometry, Cell biology, embryonic structures, Biological Assay, biological phenomena, cell phenomena, and immunity, Microtubule-Associated Proteins, Flux (metabolism) |
الوصف: | Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells. |
تدمد: | 1554-8635 1554-8627 |
الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::53d80267598cccb13eb8df34c78babaaTest https://doi.org/10.4161/auto.6.5.12112Test |
حقوق: | OPEN |
رقم الانضمام: | edsair.doi.dedup.....53d80267598cccb13eb8df34c78babaa |
قاعدة البيانات: | OpenAIRE |
تدمد: | 15548635 15548627 |
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