*4.3.3 Fluorescent dye method*

Live or dead cells that cannot be differentiated by the light microscope can be counted after fluorescent labeling. **Table 2** showed the commonly used dyes that


**Table 2.** *Summary of fluorescent dye.* label the cellular components such as nucleic acid, cytoplasm, cell membrane, redox environment, and lipase. For example, SYTO series nucleic acid fluorescent dyes, etc. stain the DNA or RNA of the live or dead cells; PI nor SYTOX Green nucleic acid dyes cannot transfer into the live cells and stain the DNA of the damaged cell membranes. Both types of dyes can be used in combination to measure the ratio of live and dead cells. After labeling, the cells can be detected by a fluorescence microscopy or by a flow cytometry. The combination of fluorescent dyes and advanced instruments makes it possible to realize the "visualization" and investigate the mechanisms of action under the physiological and pathological conditions and to explain the significance of life effects, which is of great significance in the field of disease diagnosis and drug screening.

#### **5. Drug-induced proliferation assays**

A drug is a substance that induces functional changes in an organism through chemical or physical actions, regardless of whether the resulting effect is beneficial or detrimental to the health of the receiving organism. Therefore, assays are critical in revealing interactions between the drug and the organism. In particular, the cell proliferation assays provide valuable information for exploring the pathogenesis of the disease, diagnosing the disease, and treating the disease.

Traditionally, a dose–response curve is used to evaluate the potency of an inhibitor (IC50). The commonly used cell proliferation assays are either metabolic activity-based methods such as the tetrazo-based cellular NAD(P)H detection system (MTT, CCK8 method) or the cellular ATP detection system (CellTiter-Glo Assay). According to the mechanism of metabolic activity detection, these endpoint colorimetric and fluorescence methods can be divided into five categories (**Table 3**): the reducing environment of live cells, ATP of live cells, detecting products released by dead cells, esterase, and detecting mitochondrial metabolism of live cells.

#### **5.1 Reducing environment of live cells**

Resazurin and its derivative C12-resazurin are not fluorescent, but their reduced forms are fluorescent. Dehydrogenase reduces the non-fluorescent blue resazurin into a strongly fluorescent pink resorufin [61] in enzyme- or cell-based assays. Since the dehydrogenase level is high in active cells and very low in damaged or inactive cells, the resazurin assay shows a strong signal in metabolically active cells. Interestingly, even though resazurin can be reduced by mitochondrial enzymes, no evidence of resazurin reduction was found in mitochondria as shown by confocal microscopy analysis [62].

Resazurin is water-soluble and stable in culture medium. Single reagent addition allowed for simple assays of cell viability which is especially suitable for automated manipulation and high-throughput analysis [63]. However, the fluorescence is bleached by light, so it is not suitable to track the cell growth.

#### **5.2 ATP production in live cells**

Adenosine triphosphate (ATP) is an indicator of active live cells, and its cellular level directly reflects the number and state of cells. The CellTiter-Glo method is a luminescence-based endpoint assay, commonly used for ATP measurement after cell lysis. The assay is based on ATP consumption by luciferase to produce light (maximum emission wavelength ~560 nm at pH 7.8) [64]. Due to the absence of interference of endogenous luciferase in mammalian cells, a stable glow-type signal

**47**

**5.3 Lactonase in living cells**

by 50% as analyzed by flow cytometry [66].

sity. Also, it can be used to trace in vivo studies [68, 69].

*Cell Growth Measurement*

Reducing environment of live cells

ATP production in live cells

Esterase of live cells

Released products of dead

Mitochondrial dehydrogenase of live cells

cells

**Table 3.**

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

**Classification Method Description Cell** 

Resazurin Reduced by cytosolic

CellTiter-Glo Luciferase consumes ATP

LDH release LDH released by damaged

51Cr release 51Cr released by damaged

assay

Tetrazolium Reduced by mitochondrial

formazan

FDA;calcein-AM Cleaved by esterase in

dehydrogenase in metabolically active cells to produce pink strong fluorescent resorufin

to produce light

living cells to produce a fluorescent substance retained in the cell

or dead cells reduces the tetrazolium salts to colored

or dead cells can be measured by a radioactivity

dehydrogenase to produce colored formazan products **viability**

Lysed cells

Live cells Growth

Live cells Division

Dead cells Death

Dead cells Death

Live cells Growth

**Application**

Growth

generated using luciferase can be detected as little as 0.1 picomoles ATP by luminometers, showing high sensitivity, although the signal decreases after 10–30 min. The luciferin-luciferase bioluminescence assay has been used to detect small amount of bacterial contamination in samples such as blood, milk, urine, soil, and sludge [65]. In addition, the assay can evaluate antibiotic effects, determine cell proliferation and cytotoxicity in both bacterial and mammalian cells [64], and distinguish the cytostatic and killing potential of anticancer drugs for malignant cell growth. Furthermore, it has been used for bioactive factor activity assays, large-scale antitumor drug screening, cytotoxicity assays, and tumor radiosensitivity assays.

*Comparison of commonly used methods for drug-induced proliferation assays.*

Lactonase is a non-specific esterase and cleaves a non-fluorescent molecular probe to produce a fluorescent substance. Lactonase activity is high in live cells but low in dead cells, so the fluorescence is seen and retained in live cells. In addition, the fluorescence can be evenly distributed to the two daughter cells after cell division, although each successive passage decreased the fluorescence intensity in cells

The most commonly used fluorescent probes are fluorescein diacetate (FDA) and its derivative succinimidyl ester of carboxyfluorescein diacetate, commonly known as CFSE-SE [67]. CFDA-SE was initially used for lymphocyte proliferation testing in 1994 and then applied to detect monocytes, fibroblasts, etc. The CFDA-SE method determines the cycle number of cell divisions based on fluorescence inten-

In addition, calcein-AM, (3′,6′-Di(O-acetyl)-4′,5′-bis[N,N-bis(carboxymethyl) aminomethyl] fluorescin tetraacetoxymethyl ester), also known as calcein acetyl



#### **Table 3.**

*Cell Growth*

field of disease diagnosis and drug screening.

**5. Drug-induced proliferation assays**

**5.1 Reducing environment of live cells**

microscopy analysis [62].

**5.2 ATP production in live cells**

the disease, diagnosing the disease, and treating the disease.

bleached by light, so it is not suitable to track the cell growth.

label the cellular components such as nucleic acid, cytoplasm, cell membrane, redox environment, and lipase. For example, SYTO series nucleic acid fluorescent dyes, etc. stain the DNA or RNA of the live or dead cells; PI nor SYTOX Green nucleic acid dyes cannot transfer into the live cells and stain the DNA of the damaged cell membranes. Both types of dyes can be used in combination to measure the ratio of live and dead cells. After labeling, the cells can be detected by a fluorescence microscopy or by a flow cytometry. The combination of fluorescent dyes and advanced instruments makes it possible to realize the "visualization" and investigate the mechanisms of action under the physiological and pathological conditions and to explain the significance of life effects, which is of great significance in the

A drug is a substance that induces functional changes in an organism through chemical or physical actions, regardless of whether the resulting effect is beneficial or detrimental to the health of the receiving organism. Therefore, assays are critical in revealing interactions between the drug and the organism. In particular, the cell proliferation assays provide valuable information for exploring the pathogenesis of

Traditionally, a dose–response curve is used to evaluate the potency of an inhibitor (IC50). The commonly used cell proliferation assays are either metabolic activity-based methods such as the tetrazo-based cellular NAD(P)H detection system (MTT, CCK8 method) or the cellular ATP detection system (CellTiter-Glo Assay). According to the mechanism of metabolic activity detection, these endpoint colorimetric and fluorescence methods can be divided into five categories (**Table 3**): the reducing environment of live cells, ATP of live cells, detecting products released

by dead cells, esterase, and detecting mitochondrial metabolism of live cells.

Resazurin and its derivative C12-resazurin are not fluorescent, but their reduced forms are fluorescent. Dehydrogenase reduces the non-fluorescent blue resazurin into a strongly fluorescent pink resorufin [61] in enzyme- or cell-based assays. Since the dehydrogenase level is high in active cells and very low in damaged or inactive cells, the resazurin assay shows a strong signal in metabolically active cells. Interestingly, even though resazurin can be reduced by mitochondrial enzymes, no evidence of resazurin reduction was found in mitochondria as shown by confocal

Resazurin is water-soluble and stable in culture medium. Single reagent addition allowed for simple assays of cell viability which is especially suitable for automated manipulation and high-throughput analysis [63]. However, the fluorescence is

Adenosine triphosphate (ATP) is an indicator of active live cells, and its cellular level directly reflects the number and state of cells. The CellTiter-Glo method is a luminescence-based endpoint assay, commonly used for ATP measurement after cell lysis. The assay is based on ATP consumption by luciferase to produce light (maximum emission wavelength ~560 nm at pH 7.8) [64]. Due to the absence of interference of endogenous luciferase in mammalian cells, a stable glow-type signal

**46**

*Comparison of commonly used methods for drug-induced proliferation assays.*

generated using luciferase can be detected as little as 0.1 picomoles ATP by luminometers, showing high sensitivity, although the signal decreases after 10–30 min.

The luciferin-luciferase bioluminescence assay has been used to detect small amount of bacterial contamination in samples such as blood, milk, urine, soil, and sludge [65]. In addition, the assay can evaluate antibiotic effects, determine cell proliferation and cytotoxicity in both bacterial and mammalian cells [64], and distinguish the cytostatic and killing potential of anticancer drugs for malignant cell growth. Furthermore, it has been used for bioactive factor activity assays, large-scale antitumor drug screening, cytotoxicity assays, and tumor radiosensitivity assays.

#### **5.3 Lactonase in living cells**

Lactonase is a non-specific esterase and cleaves a non-fluorescent molecular probe to produce a fluorescent substance. Lactonase activity is high in live cells but low in dead cells, so the fluorescence is seen and retained in live cells. In addition, the fluorescence can be evenly distributed to the two daughter cells after cell division, although each successive passage decreased the fluorescence intensity in cells by 50% as analyzed by flow cytometry [66].

The most commonly used fluorescent probes are fluorescein diacetate (FDA) and its derivative succinimidyl ester of carboxyfluorescein diacetate, commonly known as CFSE-SE [67]. CFDA-SE was initially used for lymphocyte proliferation testing in 1994 and then applied to detect monocytes, fibroblasts, etc. The CFDA-SE method determines the cycle number of cell divisions based on fluorescence intensity. Also, it can be used to trace in vivo studies [68, 69].

In addition, calcein-AM, (3′,6′-Di(O-acetyl)-4′,5′-bis[N,N-bis(carboxymethyl) aminomethyl] fluorescin tetraacetoxymethyl ester), also known as calcein acetyl

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 under 494 mm excitation light.
