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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359696/ https://www.ncbi.nlm.nih.gov/pubmed/32357460 http://dx.doi.org/10.3390/mps3020030 |
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author | Wilkesmann, Sebastian Westhauser, Fabian Fellenberg, Joerg |
author_facet | Wilkesmann, Sebastian Westhauser, Fabian Fellenberg, Joerg |
author_sort | Wilkesmann, Sebastian |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-7359696 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-73596962020-08-07 Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells Wilkesmann, Sebastian Westhauser, Fabian Fellenberg, Joerg Methods Protoc Protocol 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. MDPI 2020-04-26 /pmc/articles/PMC7359696/ /pubmed/32357460 http://dx.doi.org/10.3390/mps3020030 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Protocol Wilkesmann, Sebastian Westhauser, Fabian Fellenberg, Joerg 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 | 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 | Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359696/ https://www.ncbi.nlm.nih.gov/pubmed/32357460 http://dx.doi.org/10.3390/mps3020030 |
work_keys_str_mv | AT wilkesmannsebastian combinedfluorescencebasedinvitroassayforthesimultaneousdetectionofcellviabilityandalkalinephosphataseactivityduringosteogenicdifferentiationofosteoblastprecursorcells AT westhauserfabian combinedfluorescencebasedinvitroassayforthesimultaneousdetectionofcellviabilityandalkalinephosphataseactivityduringosteogenicdifferentiationofosteoblastprecursorcells AT fellenbergjoerg combinedfluorescencebasedinvitroassayforthesimultaneousdetectionofcellviabilityandalkalinephosphataseactivityduringosteogenicdifferentiationofosteoblastprecursorcells |