Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon
Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systema...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-03-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/12/4/196 |
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| author | Darja Lavogina Helen Lust Maris-Johanna Tahk Tõnis Laasfeld Hans Vellama Naila Nasirova Markus Vardja Kattri-Liis Eskla Andres Salumets Ago Rinken Jana Jaal |
| author_facet | Darja Lavogina Helen Lust Maris-Johanna Tahk Tõnis Laasfeld Hans Vellama Naila Nasirova Markus Vardja Kattri-Liis Eskla Andres Salumets Ago Rinken Jana Jaal |
| author_sort | Darja Lavogina |
| collection | DOAJ |
| description | Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin. |
| first_indexed | 2024-03-09T11:05:49Z |
| format | Article |
| id | doaj.art-13156e2fff9b4926b2b1f261243f0f3e |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T11:05:49Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-13156e2fff9b4926b2b1f261243f0f3e2023-12-01T00:57:41ZengMDPI AGBiosensors2079-63742022-03-0112419610.3390/bios12040196Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application HorizonDarja Lavogina0Helen Lust1Maris-Johanna Tahk2Tõnis Laasfeld3Hans Vellama4Naila Nasirova5Markus Vardja6Kattri-Liis Eskla7Andres Salumets8Ago Rinken9Jana Jaal10Institute of Clinical Medicine, University of Tartu, 50406 Tartu, EstoniaInstitute of Clinical Medicine, University of Tartu, 50406 Tartu, EstoniaInstitute of Chemistry, University of Tartu, 50411 Tartu, EstoniaInstitute of Chemistry, University of Tartu, 50411 Tartu, EstoniaDepartment of Physiology, Institute of Biomedicine and Translational Medicine, University of Tartu, 50411 Tartu, EstoniaInstitute of Chemistry, University of Tartu, 50411 Tartu, EstoniaDepartment of Radiotherapy and Oncological Therapy, Tartu University Hospital, 50406 Tartu, EstoniaDepartment of Physiology, Institute of Biomedicine and Translational Medicine, University of Tartu, 50411 Tartu, EstoniaInstitute of Clinical Medicine, University of Tartu, 50406 Tartu, EstoniaInstitute of Chemistry, University of Tartu, 50411 Tartu, EstoniaInstitute of Clinical Medicine, University of Tartu, 50406 Tartu, EstoniaSince 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.https://www.mdpi.com/2079-6374/12/4/196resazurinAlamar Blueresorufinfluorescenceabsorbancecell viability |
| spellingShingle | Darja Lavogina Helen Lust Maris-Johanna Tahk Tõnis Laasfeld Hans Vellama Naila Nasirova Markus Vardja Kattri-Liis Eskla Andres Salumets Ago Rinken Jana Jaal Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon Biosensors resazurin Alamar Blue resorufin fluorescence absorbance cell viability |
| title | Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon |
| title_full | Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon |
| title_fullStr | Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon |
| title_full_unstemmed | Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon |
| title_short | Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon |
| title_sort | revisiting the resazurin based sensing of cellular viability widening the application horizon |
| topic | resazurin Alamar Blue resorufin fluorescence absorbance cell viability |
| url | https://www.mdpi.com/2079-6374/12/4/196 |
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