| Автор | Hanthamrongwit, M. |
| Автор | Reid, W.H. |
| Автор | Courtney, J.M. |
| Автор | Grant, M.H. |
| Дата выпуска | 1994 |
| dc.description | There is a requirement for a convenient and reliable method for evaluating the growth rate of human keratinocytes cultured on collagen-based substrates. Therefore, three methods of determining cell growth were first used to quantify the growth rate of the well-characterised L929 mouse fibroblast cell line on tissue culture plastic and the results compared. The methods used were the measurement of total cell protein, cell counting using an electronic Coulter counter and a fluorimetric assay employing 5-carboxyfluorescein diacetate (CFDA). The CFDA assay showed the highest correlation with seeding density of the L929 cells, and the lowest standard deviations. It was the most rapid and convenient method for processing large numbers of samples. Only viable cells can deacetylate the non-fluorescent CFDA to carboxyfluorescein, which is fluorescent and accumulates inside the cells. Therefore, the assay specifically quantifies only viable cells. Subsequently, this assay has been successfully applied to the measurement of human keratinocyte growth rate on collagen gels and sponges. We have demonstrated that keratinocytes grow equally well on gels and sponges, and that media containing low calcium concentrations (0.09 mM) favour rapid proliferation of keratinocytes. Our results show that the CFDA assay is an accurate, reliable and convenient method for quantifying cell growth in vitro. It is particularly valuable when growing cells on optically opaque substrata, such as collagen sponges, where growth cannot be monitored daily by microscopy. |
| Издатель | Sage Publications |
| Название | 5-Carboxyfluorescein Diacetate as a Probe for Measuring the Growth of Keratinocytes |
| Тип | Journal Article |
| DOI | 10.1177/096032719401300610 |
| Print ISSN | 0960-3271 |
| Журнал | Human and Experimental Toxicology |
| Том | 13 |
| Первая страница | 423 |
| Последняя страница | 427 |
| Аффилиация | Hanthamrongwit, M., Bioengineering Unit, University of Strathclyde |
| Аффилиация | Reid, W.H., Burns Unit, Glasgow Royal Infirmary, Glasgow G4 ONW, UK |
| Аффилиация | Courtney, J.M., Bioengineering Unit, University of Strathclyde |
| Аффилиация | Grant, M.H., Bioengineering Unit, University of Strathclyde |
| Выпуск | 6 |
| Библиографическая ссылка | Cook JA, Mitchell JBViability measurements in mammalian cell systems. Analytical Biochemistry1989; 179: 1-7. |
| Библиографическая ссылка | Rosdy M., Grisoni B., Clauss LCProliferation of normal human keratinocytes on silicone substrates. Biomaterials1991; 12: 511-17. |
| Библиографическая ссылка | Liu SC, Eaton MJ & Karasek MA Growth characteristics of human epidermal keratinocytes from new born foreskin in primary and serial cultures. In Vitro 1979 ; 15: 813-22. |
| Библиографическая ссылка | Woodley DT, Wynn KC & Q'Keefe EJ Type IV collagen and fibronectin enhance human keratinocyte thymidine incorporation and spreading in the absence of soluble growth factors. Journal of Investigative Dermatology 1990; 94: 139-43. |
| Библиографическая ссылка | Reinwald JG & Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells Cell 1975; 6: 331-44. |
| Библиографическая ссылка | Boyce ST & Ham RG Normal human epidermal keratinocytes. In: In Vitro Model for Cancer Research, Vol III, Webber MM (ed), CRC Press, Florida , 1986, pp. 245-74. |
| Библиографическая ссылка | Smith MD, Shearer MG, Srivastava S., Scott R. & Courtney JM Quantitative evaluation of the growth of established cell lines on the surface of collagen, collagen composite and reconstituted basement membrane. Urology Research 1992; 20: 285-8. |
| Библиографическая ссылка | Smith MD, Barbenel JC, Courtney JM & Grant MH Novel quantitative methods for the determination of biomaterial cytotoxicity . International Journal of Artificial Organs 1992; 15: 191-4. |
| Библиографическая ссылка | Rotman B. & Papermaster BW Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proceedings of the National Academy of Science USA 1966; 55: 134-41. |
| Библиографическая ссылка | Schmalz G. & Netuschil L. A modification of the cell culture agar diffusion test using fluorescein diacetate staining. Journal of Biomedical Materials Research 1985; 19: 653-61. |
| Библиографическая ссылка | Lowry OH, Rosebrough NJ, Farr AL & Randall RJ Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 1951; 193: 265-275. |
| Библиографическая ссылка | Hennings H. , Michael D., Cheng C., Steinart P., Holbrook K., Yuspa H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 1980; 19: 245-254. |
| Библиографическая ссылка | Pillai S., Bikle DD, Hincenbergs M., Elias PMBiochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium. Journal of Cell Physiology1988; 134: 229-37. |
| Библиографическая ссылка | Stadler R., Detmar M., Stephanek K., Bangemann C. & Orfanos CE A rapid fluorimetric assay for determination of keratinocyte proliferation in vitro. Journal of Investigative Dermatology 1989; 93: 532-4. |
