# Resazurin-Based Assay for Quantifying Living Cells during Alkaline Phosphatase (ALP) Release
## 4 Conclusions  
ALP determination requires the real-time monitoring of cell behavior. Various cellular phenotypes are related to gene expression, which helps to understand emerging diseases including cancers. Several previous studies have shown the relationship between ALP and cell viability [38; 39]. However, those studies were conducted under the standard cell culture assay. Function endpoints of those assays are labor-intensive, time-consuming, require detachment of cells, and thus hinder real-time assays and multiplex assays. Quantitative resazurin assays of cell viability have been proposed as a rapid and inexpensive method to evaluate the viability of cells during the release of ALP, which can further be used to verify studies of cell adhesion applications such as cell-based biosensors. The main parameters affecting the results obtained from the resazurin assay include the concentration of resazurin, incubation time, and cell number. The optimal incubation time of resazurin reduction was four hours for all cells. High incubation time is more likely to reduce resorufin and form non-detectable products. The research also focused on different cells, one embryonic fibroblast cell line Balb/c3T3, and three epithelial cancer cell lines, A549, MCF-7, and Ht-29. These cells have different metabolic activities, which were noticed in this study. Ht-29 cells showed the highest activity and had 100 uM as an optimal concentration of resazurin, whereas the others had 50 uM. Optimal density corresponding to the working area is very important in cell adhesion approaches. This enhances the detection of cell viability alongside the release of ALP, which may detect intracellular processes. Cells need to be proliferated for at least four days to release detectable ALP. This time can be further increased if a study is focusing on differentiation where optimizing density is required. Figure 8: The standard curve of the absorbance values vs. alkaline phosphatase (ALP) concentration ranging from 1.5–1500 U/L was fitted by the Lineweaver–Burk model with R\({}^{2}\) = 0.99. \begin{table}
\begin{tabular}{l c c c c c} \hline \hline  & \multicolumn{4}{c}{**Quantity of ALP Release (U/L) in Each Day**} \\ \hline  & **Day 4** & **Day 5** & **Day 6** & **VC** & **Blank** \\ \hline
**Balb/c 3T3** & \((7.24\pm 0.17)\) & \((8.67\pm 0.43)\) * & \((11.67\pm 0.33)\) * & \((2.75\pm 0.10)\) * & \((0.74\pm 0.17)\) * \\
**A549** & \((10.12\pm 0.35)\) & \((12.21\pm 0.10)\) * & \((19.60\pm 0.28)\) * & \((2.84\pm 0.25)\) * & \((0.87\pm 0.33)\) * \\
**MCF-7** & \((7.97\pm 0.32)\) & \((9.51\pm 0.30)\) * & \((10.19\pm 0.25)\) * & \((3.44\pm 0.27)\) * & \((1.05\pm 0.26)\) * \\
**Ht-29** & \((19.62\pm 0.60)\) & \((23.68\pm 0.51)\) * & \((27.22\pm 0.10)\) * & \((4.27\pm 1.18)\) * & \((0.91\pm 0.32)\) * \\ \hline \hline \end{tabular}  
* After exposing cells to the corresponding optimal substrate concentration of pNPP, absorbance 405 detection was converted to unit per liter using the equation in Figure 8. The obtained data were expressed as the mean \(\pm\) SD of measurements of three independent experiments with VC (vehicle control) that had dead cells, and the blank with no cells and no substrate. \(p\)-value of post-confluence culture compared to the control (data of day 4),
* \(p\) < 0.05 is significantly different.  
\end{table} 
Table 3: Summary of ALP release from each cell line versus target days at post-confluence culture. Special attention is drawn to the morphology of cells, which was monitored and compared to the resazurin assay. The cells showed logistic growth curves where the four phases were obvious. Cells that had a longer lag phase had less confluence (about 70-80%, like MCF-7 cells). In comparison, others, which had a smaller lag phase, had a gradual log phase and thus had almost 95-100% confluence by day 4. Cell growth is also influenced by the surrounding environment (e.g., media type, serum). The recommended type of media and serum were used according to the supplier's recommendations.