**5.5 NADH and NADPH production of live cell**

NADH and NADPH are important biological cofactors for enzymes that are fundamental for various biological processes, such as energy metabolism, mitochondrial function, oxidative stress, immunological functions, and cell death [72]. Antioxidant drugs that could change cellular NAD(P)H concentrations have been effective in diseases such as aging, inflammation, neural degeneration, and cancer [73, 74]. Many tetrazolium compounds can be reduced by NADH and NADPH to produce colored formazin and have been developed commercially for cell vitality assay, as shown in **Table4** [75].

Triphenyl tetrazolium chloride (TTC) is a lipophilic and light-sensitive compound, and the TTC assay was developed back in 1894 for seed viability tests and is currently a traditional method for brain live-dead neuron evaluation after ischemic stroke [76]. MTT is the most commonly used reagent for cell proliferation or cell toxicity assays, but its formazan is water-insoluble and requires an additional DMSO solubilization step for its quantification at 540 nm [77]. WST-8 is a water-soluble tetrazolium salt, and its brown-colored formazan can be measured directly by UV absorbance at 450 nm. Because of its simple protocol, the WST-8 (CCK-8) assay has become a popular cell vitality method [78–80]. However, WST-8 is not very stable especially under the reduced condition, so EZMTT was developed for drug-induced proliferation assay [81]. EZMTT is another water-soluble tetrazolium salt which is


**49**

their utility.

*Cell Growth Measurement*

same samples.

**6. Continuous assay**

continuous assays.

**6.1 High-content analysis**

**6.2 Electrical impedance technology**

*DOI: http://dx.doi.org/10.5772/intechopen.86835*

less toxic than also, EZMTT reagent showed excellent stability and signal of background ratio. Effort has been made to develop the EZMTT to greatly enhance the

In summary, these cell metabolism-based assays are commonly used as endpoint assays for IC50 measurement to rank the potency of a drug. However, the world crisis in drug resistance in infectious disease and cancer called for a deeper look at the drug-induced proliferation assays. Since drug resistance develops owing to a small fraction of the cell population that is resistant to the drug, a sensitive method for detecting partial drug resistance is very important in preventing the occurrence of drug resistance. Another approach is to measure not only the drug potency by IC50 values but also the drug efficacy by the drug-induced proliferation rate (DIP) [15]. However, to minimize the experimental error in detecting minor growth, precise DIP rate measurement in HTS mode requires the availability of a continuous assay that can track the cell proliferation from the

Continuously tracking the cell growth is important for accurate assessment of drug effects and/or growth condition changes. Even though various endpoint assays [75, 82, 83] can be terminated at various time point to obtain the time-dependent cell proliferation curve, the procedures are labor-intensive and have high experimental error. Therefore, a nondestructive continuous assay is highly desirable and critical for the precise evaluation of drug potency and efficacy. Besides the traditional turbidimetric assay by spectrophotometry that has been used for years in microbial assays, four new technologies have been developed that can be used for

High-content analysis is a cell imaging and analysis system which includes automatic high-speed microscopic imaging, fully automatic image analysis, and data management. Through snapshot cellular microscopic imaging of a 96-well plate followed by synchronous analysis, the high-content analysis, such as the CloneSelectTM imaging system, accurately measures the cell number without any cell damage. Compared with the MTT method, the experimental deviation of dose pharmacodynamic curve obtained by CloneSelectTM imaging system is smaller and more reproducible. Therefore, high-content analysis has become a reliable choice for big pharmaceutical companies to evaluate drug antiproliferation effects in high-throughput screening, although mostly for attached mammalian cells.

Electrical impedance technology-based real-time cellular analysis (RTCA) [84–86] and Epic BenchTop optical biosensors methods [87, 88] have been used in measuring bacterial growth. However, the methods require cells to be cultured on the working electrode or a sensor array of complex structures. The methods [89] cannot detect changes in the cells themselves, so the cell damage caused by drugs and the understanding of the mechanism of drug action are difficult to assess accurately; this inevitably brings detection error and interference. In addition, the requirement of complex laboratory infrastructure further limits

sensitivity and precision of the drug-induced proliferation assay.

#### **Table 4.** *Comparison of MTT, XTT, MTS, WST-1, CCK-8, and EZMTT.*

#### *Cell Growth Measurement DOI: http://dx.doi.org/10.5772/intechopen.86835*

*Cell Growth*

under 494 mm excitation light.

**Tetrazolium Structural** 

**formula**

**Solubility (tetrazolium/ formazin)**

CCK-8/WST-8 t Soluble/soluble Yellow /orange

EZMTT o Soluble/soluble Yellow/orange

*Comparison of MTT, XTT, MTS, WST-1, CCK-8, and EZMTT.*

MTT a Soluble/insoluble Yellow/purple \*\* \*\* XTT e Soluble/soluble Yellow/orange \*\*\*\* \*\*\*\* MTS i Soluble/soluble Yellow/purple \*\* \*\*\*\* WST-1 l Soluble/soluble Yellow/orange \* \*\*\*\*

**Color (tetrazolium/ formazin)**

OD 450 nm

OD 450 nm

**Cytotoxicity Reagent** 

\* \*\*\*

Essentially nontoxic

**stability**

\*\*

**5.4 Released products from dead cells**

cytotoxicity caused by drugs, chemicals, or radiation.

**5.5 NADH and NADPH production of live cell**

ester, is a fat-soluble substance and membrane-permeable fluorescein dye [70]. It is non-fluorescent and freely enters the cytoplasm and organelles such as the mitochondrial matrix. Upon entry, calcein-AM can be hydrolyzed by intracellular esterase to produce water-soluble calcein (calcium chlorophyll), which remains in the cytosol and mitochondrial matrix and produces strong green fluorescence

The radioisotope chromium (51Cr) release method was initially developed to detect dead cells and has now gradually been replaced by the LDH release method. Due to increased membrane permeability, the damaged or dying cells release the cytosolic LDH in cell culture medium [71], so the LDH activity is proportional to the number of dead cells. The LDH release assay has been used to measure the activity of cytotoxic lymphocyte (CTL) and natural killer (NK) cells, as well as the

NADH and NADPH are important biological cofactors for enzymes that are fundamental for various biological processes, such as energy metabolism, mitochondrial function, oxidative stress, immunological functions, and cell death [72]. Antioxidant drugs that could change cellular NAD(P)H concentrations have been effective in diseases such as aging, inflammation, neural degeneration, and cancer [73, 74]. Many tetrazolium compounds can be reduced by NADH and NADPH to produce colored formazin and have been developed commercially for cell vitality assay, as shown in **Table4** [75]. Triphenyl tetrazolium chloride (TTC) is a lipophilic and light-sensitive compound, and the TTC assay was developed back in 1894 for seed viability tests and is currently a traditional method for brain live-dead neuron evaluation after ischemic stroke [76]. MTT is the most commonly used reagent for cell proliferation or cell toxicity assays, but its formazan is water-insoluble and requires an additional DMSO solubilization step for its quantification at 540 nm [77]. WST-8 is a water-soluble tetrazolium salt, and its brown-colored formazan can be measured directly by UV absorbance at 450 nm. Because of its simple protocol, the WST-8 (CCK-8) assay has become a popular cell vitality method [78–80]. However, WST-8 is not very stable especially under the reduced condition, so EZMTT was developed for drug-induced proliferation assay [81]. EZMTT is another water-soluble tetrazolium salt which is

**48**

**Table 4.**

*\*less \*\*\*\*\* the most*

less toxic than also, EZMTT reagent showed excellent stability and signal of background ratio. Effort has been made to develop the EZMTT to greatly enhance the sensitivity and precision of the drug-induced proliferation assay.

In summary, these cell metabolism-based assays are commonly used as endpoint assays for IC50 measurement to rank the potency of a drug. However, the world crisis in drug resistance in infectious disease and cancer called for a deeper look at the drug-induced proliferation assays. Since drug resistance develops owing to a small fraction of the cell population that is resistant to the drug, a sensitive method for detecting partial drug resistance is very important in preventing the occurrence of drug resistance. Another approach is to measure not only the drug potency by IC50 values but also the drug efficacy by the drug-induced proliferation rate (DIP) [15]. However, to minimize the experimental error in detecting minor growth, precise DIP rate measurement in HTS mode requires the availability of a continuous assay that can track the cell proliferation from the same samples.
