Real‐time visualization and quantitation of cell death and cell cycle progression in 2D and 3D cultures utilizing genetically encoded probes
| العنوان: | Real‐time visualization and quantitation of cell death and cell cycle progression in 2D and 3D cultures utilizing genetically encoded probes |
|---|---|
| المؤلفون: | Shankara Narayanan Varadarajan, Krupa Ann Mathew, Aneesh Chandrasekharan, Santhik Subhasingh Lupitha, Asha Lekshmi, Minsa Mini, Pramod Darvin, T. R. Santhoshkumar |
| المصدر: | Journal of Cellular Biochemistry. 123:782-797 |
| بيانات النشر: | Wiley, 2022. |
| سنة النشر: | 2022 |
| مصطلحات موضوعية: | Cell Death, Caspases, Fluorescence Resonance Energy Transfer, Apoptosis, Cell Biology, Molecular Biology, Biochemistry, Cell Division |
| الوصف: | Cancer cells grown as 3D-structures are better models for mimicking in vivo conditions than the 2D-culture systems employable in drug discovery applications. Cell cycle and cell death are important determinants for preclinical drug screening and tumor growth studies in laboratory conditions. Though several 3D-models and live-cell compatible approaches are available, a method for simultaneous real-time detection of cell cycle and cell death is required. Here we demonstrate a high-throughput adaptable method using genetically encoded fluorescent probes for the real-time quantitative detection of cell death and cell cycle. The cell-cycle indicator cdt1-Kusabira orange (KO) is stably integrated into cancer cells and further transfected with the Fluorescence Resonance Energy Transfer-based ECFP-DEVD-EYFP caspase activation sensor. The nuclear cdt1-KO expression serves as the readout for cell-cycle, and caspase activation is visualized by ECFP/EYFP ratiometric imaging. The image-based platform allowed imaging of growing spheres for prolonged periods in 3D-culture with excellent single-cell resolution through confocal microscopy. High-throughput screening (HTS) adaptation was achieved by targeting the caspase-sensor at the nucleus, which enabled the quantitation of cell death in 3D-models. The HTS using limited compound libraries, identified two lead compounds that induced caspase-activation both in 2D and 3D-cultures. This is the first report of an approach for noninvasive stain-free quantitative imaging of cell death and cell cycle with potential drug discovery applications. |
| تدمد: | 1097-4644 0730-2312 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::14b4f264cc969749a287fa851e159d88Test https://doi.org/10.1002/jcb.30222Test |
| حقوق: | CLOSED |
| رقم الانضمام: | edsair.doi.dedup.....14b4f264cc969749a287fa851e159d88 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10974644 07302312 |
|---|