Tools for Measuring Cell Proliferation Within a Population or in a Single Cell

The Drama Is In the Details

The analysis of cell proliferation is crucial for cell differentiation and cancer studies and is commonly used for evaluating compound toxicity in the drug development process. Tools for measuring cell proliferation include probes for analyzing the average DNA content and cellular metabolism in a population, as well as single-cell indicators of DNA synthesis and cell cycle–specific proteins.

Here we describe a number of assays that each provide a unique window through which to view the process of cell proliferation (Tables 1A–1E). These reagents can be used individually or together to form the basis of an assay for cell proliferation, cytotoxicity, or drug efficacy.

Determining Cell Proliferation Based on DNA Content

The CyQUANT® cell proliferation assay provides a fluorescence microplate–based method for accurately counting cells in a population, based on cellular DNA content. Because cellular DNA content is highly regulated, the CyQUANT® assay can be used at multiple time points to calculate the average proliferation rate of a cell population. Determination of cell number with the CyQUANT® assay is also a highly sensitive indicator of cytotoxicity.

Three generations of CyQUANT® assays (Table 1A) provide options for different workflows: the original CyQUANT® Kit is designed for multi-day and endpoint assays; the CyQUANT® NF Kit provides a live-cell assay; and the CyQUANT® Direct Kit (Figure 1) provides a live-cell assay configured with a background suppression dye. Binding of CyQUANT® dye to DNA is independent of metabolic state, producing consistent signal windows and fluorescence intensities across a range of conditions and cell types.

Determining Cell Proliferation Based on Metabolic Activity

The PrestoBlue™ and alamarBlue® reagents (Table 1B) are used for fluorescence- or absorption-based microplate assays that measure the reductive capacity of cells (Figure 2). These cell-permeant, resazurin-based compounds are blue in color and virtually nonfluorescent. When added directly to cells, resazurin is converted to resorufin by the reducing environment of a viable cell, turning red in color and becoming highly fluorescent. Damaged and nonviable cells—which are also likely to be non-proliferating cells—have decreased reductive capacity and thus generate a proportionally lower signal. Thus, PrestoBlue™ and alamarBlue® reagents provide a measure of relative cell number that can be used to assess the average proliferation rate of the populations when performed at multiple time points. Because of the live-cell nature of the cell proliferation assays with PrestoBlue™ or alamarBlue® reagents, you can perform downstream functional analyses that are not possible with assays that require cell lysis. Thus, these assays are extremely valuable when using cells available in limited supply, such as primary cells.

The new PrestoBlue™ reagent provides robust data in as little as 10 minutes, whereas most other resazurin-based cell viability reagents on the market require a 1- to 4-hour incubation. PrestoBlue™ reagent is the ideal choice when using cells available in limited supply, such as primary cells, or when assay times are critical. For protocols that require a traditional tetrazolium-based metabolic assay, we offer the Vybrant® MTT Cell Proliferation Assay Kit.

Figure 2. Cell number determination with PrestoBlue™ reagent in less time. CHO-K1 cells were plated in cell culture medium in a 384-well plate in quadruplicate starting at 10,000 cells/well with a two-fold serial dilution. Cells were incubated with PrestoBlue™ reagent, alamarBlue® reagent, or CellTiter-Blue® reagent for (left) 10 min or (right) 4 hr. Fluorescence measurements obtained in 10 min with PrestoBlue™ reagent take 4 hr with alamarBlue® and CellTiter-Blue® reagents.

Following Cell Division with CellTrace™ Stains

The amine-reactive succinimidyl ester of carboxyfluorescein diacetate (CFSE) is the most widely used probe for generational analysis of cells, including one study which reported the resolution of 8–10 successive generations of lymphocytes [1]. CFSE binds covalently to both intracellular and cell-surface proteins, resulting in more homogeneous cellular labeling and, consequently, better intergenerational resolution than other cell-tracking dyes such as the membrane marker PKH26. When cells divide, CFSE labeling is distributed equally between the daughter cells, which are therefore half as fluorescent as the parents. As a result, each successive generation in a population of proliferating cells is readily detected using a flow cytometer, fluorescence microscope, or fluorescence microplate reader (excitation/emission maxima ~495/525 nm). CFSE is available as a component of the CellTrace™ CFSE Cell Proliferation Kit (Table 1C).

Functionally analogous to CFSE, CellTrace™ Violet stain partitions equally between daughter cells during division, producing successive two-fold reductions in cell-associated fluorescence intensity. When analyzed by flow cytometry, this label partitioning provides a direct indication of cell proliferation (Figure 3). CellTrace™ Violet stain generates blue fluorescence; consequently, it can be used to analyze proliferation of GFP-expressing cells or, in combination with CFSE, to track cells from different origins after mixing.

Tracking Cell Cycle Distribution with Vybrant® DyeCycle™ Stains

Live-cell studies of DNA content and cell cycle distribution are useful for detecting variations in growth patterns, such as in the study of tumor behavior and suppressor gene mechanisms. In a given population, cells will be distributed among three major phases of the cell cycle: the G₀/G₁ phase (one set of paired chromosomes per cell), the S phase (DNA synthesis with a variable amount of DNA per cell), and the G₂/M phase (two sets of paired chromosomes per cell prior to cell division).

We offer four Vybrant® DyeCycle™ stains for flow cytometric analysis of DNA content in live cells (Table 1). The cell-permeant Vybrant® DyeCycle™ stains are nonfluorescent until bound to double-stranded DNA, and the fluorescence signal generated upon DNA binding is proportional to DNA content. Fluorescence data can then be used to generate a DNA profile histogram that reveals the phases of the cell cycle (Figure 4). For cell cycle analysis in fixed cells, we offer the FxCycle™ Violet and FxCycle™ Far Red stains (Table 1D).

Figure 4. Sorting of live-cell populations with Vybrant® DyeCycle™ Orange stain. Cells were sorted based on G₀/G₁ and G₂/M gates using a BD FACSVantage™ flow cytometer (BD Biosciences) with 488 nm excitation and a 585/42 nm bandpass filter.

Detecting Active DNA Synthesis at the Single-Cell Level

Although Vybrant® DyeCycle™ stains can be used to determine cell cycle distributions within a population, the accuracy of this measurement decreases in complex populations, such as those with polyploidy. The only way to accurately detect cells in S phase is to actually measure DNA synthesis. Like traditional [3H] thymidine and bromodeoxyuridine (BrdU) assays, the Click-iT® EdU assay (Table 1E) is based on the incorporation of a nucleoside analog into newly synthesized DNA. In this case, however, the nucleoside analog is EdU (5-ethynyl-2’-deoxyuridine) and it is detected not with radioactivity or an antibody but by a click reaction—a copper-catalyzed covalent reaction between an azide and an alkyne [2]. In the Click-iT® EdU assay, the EdU nucleoside contains the alkyne and the fluorescent detection reagent contains the azide. The Click-iT® EdU signal can be directly visualized using fluorescence microscopy (Figure 5) or flow cytometry and is especially useful for high-content screening (HCS) assays. It has proven effective in diverse species including plants, bacteria, yeast, and a variety of animals.

Visualizing Cell Cycle Progression with Premo™ FUCCI Cell Cycle Sensor

The Premo™ FUCCI Cell Cycle Sensor allows visualization of cell cycle progression in live cells using traditional fluorescence microscopy or high-content imaging. FUCCI, the fluorescence ubiquitination cell cycle indicator, is a sensor that employs GFP and RFP, each of which is fused to one of two regulators of the cell cycle: geminin and Cdt1 [3]. This cell cycle indicator exhibits a dynamic color change from red to green, marking an individual cell’s progression through the cell cycle (Figure 6). The Premo™ FUCCI Cell Cycle Sensor combines the Cdt1 and geminin FP constructs with the powerful BacMam gene delivery and expression system. Transduction with BacMam reagents is efficient and reproducible in most cell types, without apparent cytopathic effects.

Figure 6. Imaging cell cycle progression in live cells with Premo™ FUCCI Cell Cycle Sensor. (A) Schematic of cell cycle-dependent expression of the FUCCI fluorescent protein reporters. (B) U2OS cells were transduced with Premo™ FUCCI Cell Cycle Sensor. Images were collected over 15 hr.

References

1. Lyons AB (1999) Immunol Cell Biol 77:509–515.
2. Salic A and Mitchison TJ (2008) Proc Natl Acad Sci U S A 105:2415–2420.
3. Sakaue-Sawano A, Kurokawa H, Morimura T et al. (2008) Cell 132:487–498.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.