# Resazurin-Based Assay for Quantifying Living Cells during Alkaline Phosphatase (ALP) Release
## 1 Introduction  
Biomedical studies use cell adhesion approaches in their applications (e.g., on sensors or on lab-on-a-chip [1; 2]). Simplifying the determination of cell viability on these applications is important. The common membrane integrity assay [3] that is used widely requires cell lysis and results in an endpoint assay [4]. Metabolic based assays are simple, cheap, and are a one-step assay, thereby avoiding the limitations of the above-mentioned assay. Numerous dyes can be used to reflect metabolic activity through redox reactions in live cell membranes. One of these dyes is resazurin, which under constant experimental conditions, can provide a stable rate of fluorescence intensity and quantify cell numbers [5; 6; 7]. Resazurin can be defined as a cell permeable redox indicator that helps in tracking the number of viable cells. Physiological buffers can absorb resazurin, resulting in a deep blue solution. Viable cells have active metabolisms and can therefore convert resazurin into a pink fluorescent solution [8]. Figure 1 shows a schematic of an optimization protocol for quantifying cell number by the resazurin assay. Different concentrations of resazurin are added to the culture medium of incubated cells. At various time intervals, the redox reaction, in which resazurin is reduced by the cells, is measured by fluorescence readings at 544 nm and 590 nm. The optimal concentration of rezaurin versus particular cell type is used at different cell numbers to facilitate the relationship between the fluorescent signal and the cell number at constant time. Resazurin is more sensitive and allows multiple assays compared to other dyes [9].  
Several reports have shown the optimization of resazurin reduction assays [10] for cytotoxic agents, antifungal agents, antibacterial agents, the identification of parasites, and the drug sensibility of parasites for large-scale screenings, biofilm, and for cell expansion in matrix scaffolds using bacteria [11,12] fungi and yeast [13, 14, 15], parasites [16, 17], and human cells [15, 18]. To the best of our knowledge, this is the first paper that discusses cell viability during cancer biomarker release using a resazurin-based assay. Alkaline phosphatase, which is the early biomarker of many cancers, is correlated with cell proliferation, differentiation, and apoptosis [19, 20, 21, 22, 23, 24, 25], and, therefore, is important in cell-based biosensors [26]. Cell viability was determined for cancer biomarkers using membrane integrity [27], flow cytometry [28], and phase-contrast microscope images [29]. The aim of this paper was to provide simple and reproducible methods for quantifying cell number during ALP release from different cell types. The cancer cell lines including MCF-7, A549, and Ht-29 are used as models of the top three cancers diagnosed worldwide: lung (13%), breast (11%), and colon (10%), respectively [30]. In addition, Balb/c 3T3, which is an embryonic cell line, is used as it is more sensitive to contact inhibition of cell division than cancer cell lines. The ALP level is investigated using optical methods, and the cell number is verified using the hemocytometer assay.