There are many methods and kits currently available that are used to measure numerous aspects of cell proliferation, mitochondrial function and, indirectly, cell viability. However, over-analysis of these assays and incorrect interpretation of the information they provide are becoming more frequent in the published literature. In a cell proliferation assay, the output should give you a direct and accurate measurement of the number of actively dividing cells in a population, be it cells in culture or tissues. In contrast, a cell viability assay is designed to provide an indication of the number of “healthy” cells within a population, frequently by assessing specific indicators of metabolically active cells, often related to mitochondrial function. Unlike a proliferation assay, these measurements do not distinguish between quiescent/senescent and actively dividing cells. Beta-galactosidase activity assay or staining can indicate senescent cells [1]. Assays for the cell cycle analysis and cell death are discussed elsewhere.

Label-free approaches, such as xCELLigence RTCA SP/DP Real Time Cell Analyzer from ACEA Biosciences, can measure the increase in cell numbers, and thus quantify the cell adhesion, proliferation, detachment/death [2, 3].

Figure 1. Method for measuring BrdU incorporation

The traditional method for assaying cell proliferation is to measure DNA synthesis by assessing the incorporation of a labeled DNA analog or precursor (5-bromo-2’-deoxyuridine (BrdU [4] ), an analog of pyrimidine which gets incorporated it to new DNA in the place of thymidine, or [3H]-thymidine) into the genomic DNA of cells during S phase of the cell cycle. For example, Maun HR et al assessed the proliferation of bronchial smooth muscle cells with 3H-thymidine incorporation [5]. For cells in culture, the most common method is to assess BrdU incorporation by colorimetric ELISA (Figure 1). Similar methods can be used to assess proliferating cells in vivo by pulse-labeling tissues with BrdU before harvesting, followed by assessing BrdU by ELISA or immunohistochemical staining. This can also be assessed by flow cytometry; an example trace of BrdU vs. 7-AAD is shown in Figure 2.

A newer approach is to incorporate the alkyne-containing thymidine analog EdU (5-ethynyl-2´-deoxyuridine) into DNA and detect the incorporated EdU by a click reaction—a copper-catalyzed azide–alkyne cycloaddition [6] - in the case of labeling in live animals, EdU can be either injected or mixed in the drinking water [7]. Ouadah Y et al injected, i.p., BrdU and Edu daily into mice and detected the labelled cells through an anti-BrdU antibody (Abcam ab6326) and Click-iT EdU kits from Thermo Fisher on the cryosections to investigate the activation of pulmonary neuroendocrine cells [8]. A Marconi et al pulse-chased chondrogenesis in embryonic and adult skate, Leucoraja erinacea through EdU labeling and detection with Click-iT EdU Cell Proliferation Kit from Thermo Fisher [9]. Proliferating EdU+ cells can also be detected through flow cytometry with the Click-iT Plus EdU Flow Cytometry Assay Kit from Thermo Fisher [10].

Figure 2. An example FACS trace of BrdU vs. 7-AAD. Courtesy and © Becton, Dickinson and Company. Reprinted with permission.

BrdU itself may affect the cell physiology. It can promote transcription factor- and chemical-induced reprogramming [11] and may also alter DNA structure [12].

In sections of fixed animal tissues and cell populations, proliferation is usually assessed by immunostaining for specific proliferative markers, some of which are described here. Ki-67 is a nuclear protein associated with cell proliferation and ribosomal RNA transcription [13] and commonly used [14]. Traditional Ki67 antibodies are limited in that they can only be used to stain frozen, and not paraffin-embedded sections. However, novel MIB-1 antibodies, directed against a different epitope of Ki67 can also be used to stain formalin and paraffin-fixed sections, increase the application of Ki67 as a proliferative marker. Ouadah Y et al stained mouse lung cryosections with Ki-67 antibodies (Thermo Fisher 41-5698-82 and 50-5698-82) to investigate injury-induce neuroendocrine cell proliferation [8]. Nam S et al used Ki-67 staining and flow cytometry to estimate fraction of cells from 3D hydrogel culture in G0 and G1 phases of the cell cycle [15]. Hu CK et al, on the other hand, stained cells at the G2/M transition of the cell cycle in killifish embryos with Hoechst and antibodies to phospho-Histone H3 (Ser10) to assess cell proliferation [16].

Proliferating cell nuclear antigen (PCNA) is another commonly used marker of cell proliferation. It expedites DNA synthesis with DNA polymerase-δ by encircling the genome facilitating replication by holding the polymerase to the DNA. It is therefore expressed in the nucleus during DNA synthesis and as such, can be used as a marker of cell proliferation. It also plays important roles in DNA repair. Other proliferating markers include MCM2 [17].

All assays either directly or indirectly measuring DNA synthesis are intrinsically sensitive to the stage of the cell cycle. Depending on the outcome of the assay, it may, therefore, be necessary to synchronize the cells, either by serum withdrawal which accumulates cells in G1 (which may also affect viability) or chemically inhibit DNA synthesis, blocking cells in S phase, with thymidine, cytosine arabinoside, hydroxyurea or aminopterin. In addition, cell proliferation can also be measured through monitoring of cell division by using, for example, Cell Proliferation Dye eFluor™ 670 from Thermo Fisher [18].

Cell-based assays to measure viability can be divided mainly into three categories: those that exploit the loss of membrane integrity, those that directly measure metabolic markers, and those that assess metabolic activity. Other forms of detection exist. Crystal violet staining can check the adherence of cells and thus measure the viability of adherent cells [19, 20]. For example, Lee YR et al used crystal violet staining to assess the effects of PTEN K342/K344R mutant on the proliferation of PC3 cells [21]. Vasan N et al stained fixed MCF10A cells with crystal violet to measure cell proliferation [22].

There are now many different metabolic assays available, many of which are discussed below. These assays either involve measuring levels of important metabolic proteins such as ATP or utilize the reduction of either tetrazolium salts or resazurin dyes inside cells. The ratios of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD all increase during cellular proliferation. In the presence of these metabolic intermediate cellular dehydrogenases or reductases, tetrazolium salts get reduced to a formazan product, which can be detected by the resulting colorimetric change. Similarly, resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a non-fluorescent blue redox dye that gets reduced to resorufin, a red fluorescent compound, giving both fluorescent and colorimetric changes. Commonly used substrates, together with some of their pros and cons, are described here:

• MTT: MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) is a tetrazolium salt that gets reduced by both mitochondrial and extra-mitochondrial dehydrogenases to form insoluble blue formazan crystals, meaning a solubilization step is required before the assay can be read [23]. In addition, cells become non-viable during this assay, meaning that repeat or complementary assays cannot be carried out on the same plate of cells.
• MTS/XTT: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) substrates are similar to MTT. However, one advantage is that the reactions are carried out intracellularly in the presence of the intermediate electron acceptor phenazine methosulfate (PMS), which enhances their sensitivity. In addition, the reduced formazan product is soluble and gets released to the culture media, removing the need for the extra solubility step that is required with MTT. However, phenol red in cell culture media, fatty acids, and serum albumin have all been reported to distort data obtained from MTS, XTT, and WST assays over prolonged incubation periods [24]. Promega RealTime Glo MT Cell Viability Assay explores the reductive environment inside cells to reduce a chemical into a suitable substrate for NanoLuc luciferase. Reynders M et al, for example, measured cell cytotoxicity and proliferation with an MTS assay [25].
• Alamar Blue: Alamar Blue is a resazurin compound that gets reduced to resorufin and dihydroresorufin in viable cells. It can enter live cells so does not require cell lysis, and is stable in culture media. This assay has the added advantage that it can be measured in both fluorimetric and colorimetric plate readers. Silva MC et al used Thermo Fisher Alamar Blue reagent to measure the cell viability of induced pluripotent cell-derived neural progenitor cells upon treatment of tau protein degrader QC-01–175 [26]. Dominy SS et al measured the viability of human neuroblastoma SH-SY5Y cells challenged with Porphyromonas gingivalis with or without gingipain inhibitors or antibiotics using Thermo Fisher Alamar Blue reagent [27].
• WST: Water-soluble tetrazolium salts (WSTs) are cell-impermeable tetrazolium dyes that get reduced extracellularly via plasma membrane electron transport [28], and combined with the electron acceptor PMS to generate water-soluble formazan dyes. Noda S et al, for example, examined the viability of primary dental pulp stem cells with Cell Counting Kit-8 (containing WST-8 solution, also called CCK-8 assay), from Dojindo Laboratories [29]. L Zhao et al used the same kit to measure tumor cell proliferation in culture [30]. Luther A et al used a similar WST-8 kit, but from MilliporeSigma, to measure the cytotoxicity of prospective antibiotics on HeLa and HEP G2 cells [31].

There are numerously available assays that measure ATP levels as an output of overall cell health. When cells begin to undergo apoptosis or lose membrane integrity, ATP stocks become depleted through the activity of ATPases that concurrently prevent any new ATP synthesis. This leads to a rapid depletion of intracellular ATP levels. Luminescent ATP assays (such as Promega’s CellTiter-Glo) function by lysing cells to release ATP stores, while concurrently inhibiting ATPases. Luciferase catalyzes the oxidation of luciferin to oxyluciferin in the presence of magnesium and ATP, resulting in a luminescent signal that directly correlates with the intracellular ATP concentration [32, 33].

There are a number of decisions to make when selecting the appropriate metabolic assay for your needs. Each of the substrates listed above, and related ones not covered here, have their distinct advantages and disadvantages when directly compared. Assay sensitivity, noise to signal ratio, ease of use, and reagent stability are all factors to consider. An additional important consideration with metabolic assays is that the reduction of these substrates is impacted by changes in intracellular metabolic activity that have no direct effect on overall cell viability; therefore the question you are trying to answer will play a key role in selecting the appropriate assays.

All viability assays in this category rely on the breakdown of the cell membrane during the loss of viability to either allow macromolecules to enter the cell, or allow intracellular proteins to be secreted to the culture media.

• LDH: Lactate dehydrogenase is a ubiquitous, stable cytoplasmic enzyme that converts lactate to pyruvate. If the cell membrane has been damaged, LDH, and therefore, its enzymatic activity is released from cells and can be detected in cell culture media. During the conversion of lactate to pyruvate, NAD+ gets reduced to NADH/H+. LDH-based viability assays capitalize on the formation of the free hydrogen ion by catalyzing the transfer of H+ from NADH/H+ to the tetrazolium salt INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride), reducing it to a red formazan dye [34], or conversion of resazurin to the fluorescent form resorufin in Promega CytoTox-ONE assay [35]. Persson EK et al used Pierce LDH cytotoxicity assay kit to measure necrotic cell death due to Charcot-Leyden crystals [36].
• Trypan Blue: Staining a cell suspension with trypan blue is one of the oldest and simplest viability assays [37, 38]. In a healthy, viable cell, the intact membrane will prevent trypan blue from entering cells. In dead or dying cells, trypan blue will enter the cell, staining it blue. This method was traditionally quantified manually using microscopes and hemocytometers, making it very labor-intensive. However, the recent availability of affordable automated cell counters, for example, Bio-Rad TC20 automated cell counter [38] or Vi-CELL™ automated cell viability analyzer and counter from Beckman Coulter [39], makes this assay less time consuming and more accurate than previously. Other similar, but fluorescent dyes can be used as well. For example, Aizarani N et al identifed viable cells with a cell sorter through Zombie Green from BioLegend [40].
• Calcein-AM: Calcein-acetoxymethylester is a non-fluorescent dye that is used in both cell viability and apoptosis assays, for example, for detecting the cytotoxicity of Abeta peptide and its oligomers [41]. It is lipophilic, allowing easy passage through the cell membrane. Once inside the cell, intracellular esterases cleave the ester bonds of the acetomethoxy group, resulting in the formation of a fluorescent anionic and hydrophilic calcein dye, which gets trapped inside the cell. Non-viable cells do not contain active esterases, allowing this assay to be used as a measure of viability. Cu²⁺, Co²⁺, Fe³⁺, Mn²⁺, and Ni²⁺ quench the fluorescent signal from calcein at physiological pH, which means care must be taken to select the appropriate cell culture media.
• Propidium Iodide/7-AAD: These intercalating agents are frequently used to study the cell cycle as discussed above. However, since they are membrane-impermeable, they are excluded from viable cells. This means that the fluorescence signal emitted by PI or 7-AAD in non-viable cells can be measured either by fluorescence microscopy [42] or FACS analysis [43, 44]. For example, Nortley R et al evaluated the pericyte cell death with 7.5 uM propidium iodide [45]. Capello M et al used IncuCyte™ Cytotox Green Reagent from Essen Bioscience to measure the cell death among cultured cancer cells [46].
• Cell-impermeable DNA-binding dyes such as DRAQ7 from Abcam [47] or SYTOX from Thermo Fisher. These dyes enter cells through compromised cell memebranes and display strong fluorescence upon binding with DNA. For example, Samir P et al monitored cell death in cultured bone-marrow-derived macrophages in real time with SYTOX Green staining under a two-colour IncuCyte Zoom incubator imaging system from Essen Biosciences [48].

This section is provided by Labome to help guide researchers to identify most suited cell proliferation and cell viability assay kits. Labome surveys formal publications. Table 2 lists the major suppliers for reagents/kits used in the cell-based assays. Some of the applications are enumerated here. Zeng Q et al measured breast cancer cell growth with an MTT cell proliferation kit from Roche [49]. Nam S et al measured cell proliferation in 3D-hydrogel with EdU from Thermo Fisher Scientific [15]. Ombrato L et al assessed the in vitro proliferation of MMTV–PyMT actin–GFP cells in 2D co-culture with MACS-sorted EPCAM+ and Ly6G+ mouse cells using Click-iT Plus EdU Flow Cytometry Assay Kit from Thermo Fisher in a flow cytometer [50]. Silvestre-Roig C et al assayed the cell viability of smooth muscle cells incubated with isolated NETs with propidium iodide, Vybrant MTT cell proliferation assay from Thermo Fisher, and for live imaging, calcein AM from Thermo Fisher [51]. Genet G et al measured the proliferation of HUVEC cells in response to VEGF-A with the xCELLigence RTCA DP analyzer from ACEA Biosciences [3]. Herb M et al assessed the viability of macrophages in culture with the CyQUANT Direct Cell Proliferation Assay from Thermo Fisher Scientific [52].

This article is derived from an earlier version of an article authored by Dr. Laura Cobb "Cell-Based Assays: the Cell Cycle, Cell Proliferation and Cell Death", written in February 2013.