# Resazurin-Based Assay for Quantifying Living Cells during Alkaline Phosphatase (ALP) Release
## 3 Results and Discussion
### Determining the Optimal Density of Cell Lines  
There are several stages in the life cycle of mammalian cells. In one of these stages, hormones, protein, enzymes, etc. are produced. To allow the expression of these products, cellular functions such as adhesion, proliferation, migration, or apoptosis are usually investigated [9; 10]. Cells were plated at an initial density of (\(320\times 10^{3}\) cell/mL) and then serially diluted in a ratio of 1:2 to produce the range of cell density/well. This was done to make the cells simultaneously reach contact-inhibited growth. Cell growth was monitored over five days and resazurin reduction was recorded. Figure 5A-D shows the growth curves of the four cell lines at the five different densities. Low density with an s-shape expressed logistic growth, whereas high density failed to exhibit an s-shape and levelled out from day 1. Balb/c 3T3 cell lines displayed an s-shape at \(20\)-\(40\times 10^{3}\) cells/mL, but for the higher densities, \(80\)-\(320\times 10^{3}\) cell/mL  
Figure 4: Experimental and theoretical standard curves of resazurin at various cell lines. (**A**) Balb/3T3 cell lines; (**B**) A549 cell lines; (**C**) MCF-7 cell lines; (**D**) Ht-29 cell lines; (**E**) Coefficient variance % of experimental curves vs. cells numbers. (**F**) Errors % of theoretical and experimental curves vs. cell number. Data are expressed as mean \(\pm\) SD of measurements of three independent experiments.  
showed approximately level density, although \(80\times 10^{3}\) cell/mL exhibited a little growth at day 1, which means that \(40\times 10^{3}\) cell/mL is the optimal density of the Balb/c 3T3 cell lines (Figure 5A). The A549 cell lines behaved the same as the previous cell line, but the s-shape of the lowest density \(20\times 10^{3}\) cell/mL was quite unclear. From Figure 5B, it is clear that \(40\times 10^{3}\) cell/mL is the optimal density (Figure 5B). Like the Balb/c 3T3 cell lines, the A549 cell lines at the higher densities ranged from 80-320 \(\times 10^{3}\) cell/mL and levelled by day 1. Figure 5C shows that the MCF-7 cell lines displayed an s-shape of \(20\times 10^{3}\) cell/mL, but at a very low reduction rate of resazurin (\(\sim\)1000 nm). The \(40\times 10^{3}\) cell/mL sample demonstrated a J-shape, which began to level by day 5. The higher densities, ranging from 160-320 \(\times 10^{3}\) cell/mL, did not show any growth behavior. This meant that \(40\times 10^{3}\) cell/mL was the optimal density. Ht-29 behaved quite differently to the other cell lines, where at low densities 20-40 \(\times 10^{3}\) cell/mL, it showed a j-shape (Figure 5D), whereas at \(80\times 10^{3}\) cell/mL, it showed an s-shape, which makes it the optimal density. Like the other cells, the Ht-29 cell lines at the higher density ranged from 160-320 \(\times 10^{3}\) cell/mL and levelled on day 1 and then started to decrease. The CV% of all five days was less than 20% on day 1, and on day 2, cell numbers barely exceeded 20%. This was probably because of the lag phase and the ability of cells to divide and adjust themselves in the new environment. This investigation facilitated the determination of the optimal density of each cell line and makes it applicable for this research study, where the expression of ALP versus cell viability was investigated [35; 36].  
Figure 5: Logistic growth curves of the different cell densities at various cell lines over five days. **(A)** Balbç/3T3 cell lines; **(B)** A549 cell lines; **(C)** MCF-7 cell lines; **(D)** Ht-29 cell lines; **(E)** coefficient variance % of growth curves vs. cells numbers. Data are expressed as the mean \(\pm\) SD of measurements of three independent experiments.