Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells
The aim of the study was mammosphere assay optimization for quantifcation of IL6-induced stemness in differentiated (СD44– ) T47D breast cancer cells.Material and Methods. The effect of three commonly used cell-detaching methods (TrypLE, accutase, cell scrapper) at various confuence (40–50 % and 70–...
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| Format: | Article |
| Language: | Russian |
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Russian Academy of Sciences, Tomsk National Research Medical Center
2023-01-01
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| Series: | Сибирский онкологический журнал |
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| Online Access: | https://www.siboncoj.ru/jour/article/view/2375 |
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| author | K. V. Nevskaya L. V. Efimova P. V. Korda D. S. Dolgasheva M. K. Ibragimova M. E. Menyailo E. V. Udut N. V. Litviakov A. G. Pershina |
| author_facet | K. V. Nevskaya L. V. Efimova P. V. Korda D. S. Dolgasheva M. K. Ibragimova M. E. Menyailo E. V. Udut N. V. Litviakov A. G. Pershina |
| author_sort | K. V. Nevskaya |
| collection | DOAJ |
| description | The aim of the study was mammosphere assay optimization for quantifcation of IL6-induced stemness in differentiated (СD44– ) T47D breast cancer cells.Material and Methods. The effect of three commonly used cell-detaching methods (TrypLE, accutase, cell scrapper) at various confuence (40–50 % and 70–80 %) on cell viability, phenotypic profle and mammosphere formation was tested. The cell viability was examined using AnnexinV/propidium iodide assay. The phenotypic profle was analyzed by fow cytometry with fuorescent markers CD24 and CD44.Results. Detachment of the cells using scrapper led to substantial increase in early apoptotic and late apoptotic cells in comparison with TrypLE and accutase. Dissociation with TrypLE reduced the percentage of detected CD44+ positive cells, whereas accutase saved the surface marker. The number of mammosphere and their diameter did not differ between groups. Incubation of differentiated (CD44– CD24+) T47D cells with IL-6 for 24 hours resulted in an appearance of CD44+CD24+ and CD44+CD24–/low subpopulation. Furthermore, the differentiated cells after 24 hours of IL6 exposure formed 3 times more mammospheres compared to the control.Conclusion. Usage of cells with confuence of no more than 80 % and accutase for detachment of cells is recommended for mammosphere assay. Incubation of CD44– CD24+ T47D cells with IL6 for 24 hours is suffcient for stimulation of stemness plasticity. |
| format | Article |
| id | doaj-art-9f072065bc7f44e98f6df083750603d4 |
| institution | Kabale University |
| issn | 1814-4861 2312-3168 |
| language | Russian |
| publishDate | 2023-01-01 |
| publisher | Russian Academy of Sciences, Tomsk National Research Medical Center |
| record_format | Article |
| series | Сибирский онкологический журнал |
| spelling | doaj-art-9f072065bc7f44e98f6df083750603d42025-08-20T03:43:40ZrusRussian Academy of Sciences, Tomsk National Research Medical CenterСибирский онкологический журнал1814-48612312-31682023-01-01216596710.21294/1814-4861-2022-21-6-59-671063Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cellsK. V. Nevskaya0L. V. Efimova1P. V. Korda2D. S. Dolgasheva3M. K. Ibragimova4M. E. Menyailo5E. V. Udut6N. V. Litviakov7A. G. Pershina8Siberian State Medical University of the Ministry of Health of RussiaSiberian State Medical University of the Ministry of Health of RussiaSiberian State Medical University of the Ministry of Health of RussiaCancer Research Institute, Тomsk National Research Medical Center, Russian Academy of SciencesCancer Research Institute, Тomsk National Research Medical Center, Russian Academy of Sciences; Siberian State Medical University of the Ministry of Health of RussiaCancer Research Institute, Тomsk National Research Medical Center, Russian Academy of SciencesSiberian State Medical University of the Ministry of Health of RussiaSiberian State Medical University of the Ministry of Health of Russia; Cancer Research Institute, Тomsk National Research Medical Center, Russian Academy of SciencesSiberian State Medical University of the Ministry of Health of RussiaThe aim of the study was mammosphere assay optimization for quantifcation of IL6-induced stemness in differentiated (СD44– ) T47D breast cancer cells.Material and Methods. The effect of three commonly used cell-detaching methods (TrypLE, accutase, cell scrapper) at various confuence (40–50 % and 70–80 %) on cell viability, phenotypic profle and mammosphere formation was tested. The cell viability was examined using AnnexinV/propidium iodide assay. The phenotypic profle was analyzed by fow cytometry with fuorescent markers CD24 and CD44.Results. Detachment of the cells using scrapper led to substantial increase in early apoptotic and late apoptotic cells in comparison with TrypLE and accutase. Dissociation with TrypLE reduced the percentage of detected CD44+ positive cells, whereas accutase saved the surface marker. The number of mammosphere and their diameter did not differ between groups. Incubation of differentiated (CD44– CD24+) T47D cells with IL-6 for 24 hours resulted in an appearance of CD44+CD24+ and CD44+CD24–/low subpopulation. Furthermore, the differentiated cells after 24 hours of IL6 exposure formed 3 times more mammospheres compared to the control.Conclusion. Usage of cells with confuence of no more than 80 % and accutase for detachment of cells is recommended for mammosphere assay. Incubation of CD44– CD24+ T47D cells with IL6 for 24 hours is suffcient for stimulation of stemness plasticity.https://www.siboncoj.ru/jour/article/view/2375breast cancermammosphere formation assayil6stemness |
| spellingShingle | K. V. Nevskaya L. V. Efimova P. V. Korda D. S. Dolgasheva M. K. Ibragimova M. E. Menyailo E. V. Udut N. V. Litviakov A. G. Pershina Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells Сибирский онкологический журнал breast cancer mammosphere formation assay il6 stemness |
| title | Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells |
| title_full | Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells |
| title_fullStr | Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells |
| title_full_unstemmed | Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells |
| title_short | Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells |
| title_sort | optimization of mammosphere formation assay for quantification of il6 induced stemness in differentiated breast cancer cells |
| topic | breast cancer mammosphere formation assay il6 stemness |
| url | https://www.siboncoj.ru/jour/article/view/2375 |
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