Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells

Abstract

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.

Effect of manganese, zinc, and copper on the biological and osteogenic properties of mesoporous bioactive glass nanoparticles

“…Cell viability and ALP activity as early marker of osteogenic differentiation were determined in a combined fluorescence‐based assay. The protocol itself and the proof of high correlation between ALP activity data obtained with the presented and the acknowledged para‐nitrophenylphosphate assay were published by our group recently 34 …”

Impact of Zinc- or Copper-Doped Mesoporous Bioactive Glass Nanoparticles on the Osteogenic Differentiation and Matrix Formation of Mesenchymal Stromal Cells

“…Cell viability and alkaline phosphatase (ALP) activity as a marker of cellular osteogenic differentiation were assessed using a combined fluorescence-based assay following a previously published protocol [ 26 ]. Since it correlates with cell number and viability [ 27 , 28 , 29 ], metabolization of fluorescein diacetate (FDA) was quantified to determine cell viability.…”

“…Since it correlates with cell number and viability [ 27 , 28 , 29 ], metabolization of fluorescein diacetate (FDA) was quantified to determine cell viability. The conversion of 4-methylumbelliferyl phosphate (4-MUP), an ALP substrate, was measured as it correlates directly with ALP activity [ 26 ]. Following removal of CCM, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Life Technologies; pH 7.0–7.3), then FDA substrate solution (0.1 mg/mL FDA (Sigma-Aldrich) in acetone (Carl Roth, Karlsruhe, Germany) 1:50 diluted in DPBS) was added.…”

Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process

“…Briefly, The ALP method employs alkaline phosphatase that catalyzes the transphosphorylation of pnitrophenyiphosphate (p-NPP) to p-nitrophenol (p-NP). The change in absorbance at 405nmm due to the formation of p-NP is directly proportional to the ALP activity 37 , 38 .…”