Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells
Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an oste...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-04-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/3/2/30 |
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| author | Sebastian Wilkesmann Fabian Westhauser Joerg Fellenberg |
| author_facet | Sebastian Wilkesmann Fabian Westhauser Joerg Fellenberg |
| author_sort | Sebastian Wilkesmann |
| collection | DOAJ |
| description | Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein–diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells. |
| first_indexed | 2024-03-10T20:13:16Z |
| format | Article |
| id | doaj.art-b225fc9042a0420d9cbd61c133cfdcb6 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-10T20:13:16Z |
| publishDate | 2020-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj.art-b225fc9042a0420d9cbd61c133cfdcb62023-11-19T22:45:42ZengMDPI AGMethods and Protocols2409-92792020-04-01323010.3390/mps3020030Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor CellsSebastian Wilkesmann0Fabian Westhauser1Joerg Fellenberg2Center for Orthopedics, Trauma Surgery and Paraplegiology, University of Heidelberg, 69118 Heidelberg, GermanyCenter for Orthopedics, Trauma Surgery and Paraplegiology, University of Heidelberg, 69118 Heidelberg, GermanyCenter for Orthopedics, Trauma Surgery and Paraplegiology, University of Heidelberg, 69118 Heidelberg, GermanyNovel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein–diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells.https://www.mdpi.com/2409-9279/3/2/30osteogenic differentiationalkaline phosphatasehuman mesenchymal stromal cellosteoblast precursor cell |
| spellingShingle | Sebastian Wilkesmann Fabian Westhauser Joerg Fellenberg Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells Methods and Protocols osteogenic differentiation alkaline phosphatase human mesenchymal stromal cell osteoblast precursor cell |
| title | Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells |
| title_full | Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells |
| title_fullStr | Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells |
| title_full_unstemmed | Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells |
| title_short | Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells |
| title_sort | combined fluorescence based in vitro assay for the simultaneous detection of cell viability and alkaline phosphatase activity during osteogenic differentiation of osteoblast precursor cells |
| topic | osteogenic differentiation alkaline phosphatase human mesenchymal stromal cell osteoblast precursor cell |
| url | https://www.mdpi.com/2409-9279/3/2/30 |
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