# Resazurin-Based Assay for Quantifying Living Cells during Alkaline Phosphatase (ALP) Release
## 3 Results and Discussion
### Alkaline Phosphatase Release from Cells at Post-Confluence Culture  
The standard curve of ALP was applied in order to convert absorbance values to an enzyme unit (e.g., U/L). Figure 8 shows that the absorbance values of ALP concentration ranged from (1.5-1500 U/L). This was plotted using the Lineweaver-Burk model with good linear regression of R\({}^{2}\) of 0.99 and linear equation obtained using Origin software. Monitoring of ALP release from Balb/c 3T3 cells, A549 cells, MCF-7 cells, and HT-29 cells was conducted. ALP was induced by differentiation using post-confluence culture. Cells were cultured for two more days after reaching 95-100% confluence on day 4. Table 3 summarizes the results of ALP release in the target days for each cell with the vehicle control (VC) and the blank. It is obvious that the ALP release increased gradually from day 4 to day 6. This rise was shown from all cells. Furthermore, the data of post-confluence culture (day 5 and day 6) were compared to the control (data of day 4) using an unpaired t-test. There was a significant difference between the data of ALP release from day 5 and day 6 compared to that released from day 4. Balb/c 3T3 cells had an average level of ALP of 7.24 U/L on day 4 and the value increased on day 5 and day 6 (8.67 U/L and 11.67 U/L, respectively). The value of lung cancer cells exhibited a sharp increment from day 4 to day 6, which was 10.12 U/L, 12.21 U/L, and 19.60 U/L. The breast cancer cells showed slight incensement from the fourth day to the sixth day, and that was 7.97 U/L, 9.51 U/L, and 10.19 U/L, respectively. The colon cancer cell had the highest release value of ALP (19.62 U/L) at confluence day compared to the others and also for the post-confluence day 5 and day 6 of 23.68 U/L and 27.22 U/L, respectively. Vehicle control of all cells showed less values. The cells of Balb/3T3 and A549 had 2.75 U/L and 2.84 U/L, respectively, whereas the MCF-7 and Ht-29 cells had 3.44 U/L and  
\begin{table}
\begin{tabular}{c c c c c} \hline  & **Balb/c 3T3** & **A549** & **MCF-7** & **Ht-29** \\ \hline
**Day 4** & 3091.61 \(\pm\) 28.74 & 2784.46 \(\pm\) 43.35 & (1887.34 \(\pm\) 5.36) & (2980.70 \(\pm\) 44.51) \\
**Day 5** & 2513.44 \(\pm\) 7.77 \(\lx@sectionsign\) & (1952.29 \(\pm\) 0.82) \(\lx@sectionsign\)S & (1787.48 \(\pm\) 35.99) & (2655.06 \(\pm\) 34.07) \(\lx@sectionsign\) \\
**Day 6** & 1872.74 \(\pm\) 6.72 \(\lx@sectionsign\)S & (1415.29 \(\pm\) 44.35) \(\lx@sectionsign\)S & (1407.01 \(\pm\) 66.13) \(\lx@sectionsign\)S & (2512.21 \(\pm\) 48.32) \(\lx@sectionsign\) \\
**VC** & (412.6 \(\pm\) 9.9) \(\lx@sectionsign\)S & (597.6 \(\pm\) 23.3) \(\lx@sectionsign\)S & (237.8 \(\pm\) 3.1) \(\lx@sectionsign\)S & (623.8 \(\pm\) 4.5) \(\lx@sectionsign\)S \\
**RC** & (97.2 \(\pm\) 6.3) \(\lx@sectionsign\)S & (78.7 \(\pm\) 0.9) \(\lx@sectionsign\)S & (80.5 \(\pm\) 0.3) \(\lx@sectionsign\)S & (82.7 \(\pm\) 0.7) \(\lx@sectionsign\)S \\ \hline \end{tabular} The data of the cell viability assay-based resazurin was applied for the cells at post-confluence culture. Data are expressed as mean \(\pm\) SD of the measurements of three independent experiments. VC (vehicle control,) and RC (resazurin control). Data compared to the control (data of day 4), \(\lx@sectionsign\) (\(p\)-value \(<\) 0.05 is a significant difference), \(\lx@sectionsign\)S (1 \(>\) Z-factor \(>\) 0.5 is excellent assay).  
\end{table}
Table 2: Summary of the resazurin data of each cell line versus the target days at post-confluence.  
4.27 U/L, respectively, all of which represented significant differences when compared to the control. The buffer blank of all data was very low as no substrate was added to the assay.