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.

Linearity and sensitivity of the CyQUANT® Direct assay 
Figure 1. Linearity and sensitivity of the CyQUANT® Direct assay. CHO cells were plated at densities of 0–20,000 per well in a 384-well microplate. Cells were labeled with the CyQUANT® Direct dye according to the microplate protocol. Fluorescence intensities, measured with a fluorescence microplate reader using a FITC filter set, varied linearly with respect to cell number between 40 and 20,000 cells. The inset shows the measurement range from 0 to 1,250 cells/well. The results shown represent averages of eight experiments per data point.
Table 1A. Determining Cell Proliferation Based on Total DNA Content.
Kit or reagent Ex/Em (Abs) * Stains live cells Stains fixed cells Instrument platform † Assay features Cat. No.

CyQUANT® Direct Cell Proliferation Assay Kit

508/527

Yes

 

M

An add-and-read, lysis-free cell proliferation assay designed for use with multi-well plates, making it ideal for high-throughput screening applications.

C35011
C35012

CyQUANT® NF Cell Proliferation Assay Kit

480/520

Yes

Yes

M

Live-cell assay using a cell-permeant DNA stain that eliminates the need for cell lysis and the freeze/thaw step.

C35007
C35006

CyQUANT® Cell Proliferation Assay Kit

480/520

 

Yes

M

Run multi-day and endpoint assays with the same set of standards using this frozen assay format.

C7026

* Fluorescence excitation and emission maxima in nm, with absorption maximum in parentheses where applicable. † FC = flow cytometry. FM = fluorescence microscopy. HCS = high-content screening and analysis. M = microplate assay.

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.

Cell number determination with PrestoBlue™ reagent
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.

Table 1B. Determining Cell Proliferation based  on Metabolic Activity.
Kit or reagent Ex/Em (Abs) * Stains live cells Stains fixed cells Instrument platform † Assay features Cat. No.

PrestoBlue™ cell viability reagent

•560/590
•(570)

Yes

 

M

With an incubation step as short as 10 min, PrestoBlue™ reagent saves time compared to other resazurin-based assays such as alamarBlue® reagent.

A13261
A13262

alamarBlue® reagent

•560/590
•(570)

Yes

 

M

Easily enters into live cells, eliminating the need to lyse or further process cells.

DAL1025
DAL1100

Vybrant® Cell Metabolic Assay Kit

•563/587
•(563)

Yes

 

M, FC

Provides C12-resazurin, which is better retained than resazurin, yielding brighter signals and lower detection limits.

V23110

Vybrant® MTT Cell Proliferation Assay Kit

(570)

Yes

 

M

Absorption-based tetrazolium salt assay based on the same reduction principle as the alamarBlue® and PrestoBlue™ assays.

V13154
M6494

* Fluorescence excitation and emission maxima in nm, with absorption maximum in parentheses where applicable. † FC = flow cytometry. FM = fluorescence microscopy. HCS = high-content screening and analysis. M = microplate assay.

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.

CellTrace™ Violet stain 
Figure 3. Generational analysis using CellTrace™ Violet stain. Human peripheral blood lymphocytes were harvested and stained with CellTrace™ Violet stain. The violet peaks represent successive generations of cells stimulated with mouse anti–human CD3 antibody and interleukin-2, and grown in culture for 7 days. The peak outlined in black represents cells that were grown in culture for 7 days with no stimulus.
Table 1C. Following Cell Division with Cytoplasmic Tracers.
Kit or reagent Ex/Em (Abs) * Stains live cells Stains fixed cells Instrument platform † Assay features Cat. No.

CFSE (CFDA SE)

495/525

Yes ‡

 

FC, FM, M

Amine-reactive, cell-permeant fluorescein derivative that provides homogeneous cellular labeling.

C1157

CellTrace™ CFSE Cell Proliferation Kit

492/517

Yes ‡

 

FC

Validated for generational analysis, with a protocol optimized for flow cytometry.

C34554

CellTrace™ Violet Cell Proliferation Kit

405/455

Yes ‡

 

FC

Functionally analogous to CFSE. Can be excited with violet diode lasers and multiplexed with green-fluorescent probes.

C34557

* Fluorescence excitation and emission maxima in nm, with absorption maximum in parentheses where applicable. † FC = flow cytometry. FM = fluorescence microscopy. M = microplate assay. ‡ Fixable probe. After fixation, the GFP and RFP fusions of the Premo™ FUCCI Cell Cycle Sensor can be detected with anti-GFP antibodies and anti-RFP antibodies.

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).

Vybrant® DyeCycle™ Orange stain
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.

Table 1D. Determining Cell Cycle Distribution.
Kit or reagent Ex/Em (Abs) * Stains live cells Stains fixed cells Instrument platform † Assay features Cat. No.

Vybrant® DyeCycle™ Violet stain

Vybrant® DyeCycle™ Green stain

Vybrant® DyeCycle™ Orange stain

Vybrant® DyeCycle™ Ruby stain

369/437

506/534

519/563

638/686

Yes

Yes

FC

Cell-permeant DNA-binding dyes for live-cell studies of DNA content and cell cycle distribution. Once bound to dsDNA, these dyes emit a fluorescence signal that is proportional to DNA content. Fluorescence data can be used to generate a DNA profile histogram that reveals the phases of the cell cycle.

V35003

V35004

V35005

V10309

V10273

FxCycle™ Violet stain

FxCycle™ Far Red stain

358/461

640/658

 

Yes

FC

FxCycle™ Violet (DAPI) preferentially stains dsDNA in fixed cells, whereas FxCycle™ Far Red stain requires addition of RNase A.

F10347

F10348

* Fluorescence excitation and emission maxima in nm, with absorption maximum in parentheses where applicable. † FC = flow cytometry. FM = fluorescence microscopy. HCS = high-content screening and analysis. M = microplate assay.

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.

Proliferating cells in rat ileum 
Figure 5. Proliferating cells in rat ileum. Cell proliferation was detected with the Click-iT® EdU Alexa Fluor® 594 Imaging Kit. Proliferating cells fluoresce red; nuclei are stained with the blue-fluorescent counterstain Hoechst 33342.

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.

Premo™ FUCCI Cell Cycle Sensor
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.

Table 1E. Specific Cell Cycle Markers.
Kit or reagent Ex/Em (Abs) * Stains live cells Stains fixed cells Instrument platform † Assay features Cat. No.

Click-iT® EdU Alexa Fluor® 488 Imaging Kit

Click-iT® EdU Alexa Fluor® 555 Imaging Kit

Click-iT® EdU Alexa Fluor® 594 Imaging Kit

Click-iT® EdU Alexa Fluor® 647 Imaging Kit

495/519

555/565

590/615

650/670

Yes **

 

FM

Following EdU incorporation in cells or whole animals, Click-iT® EdU staining of cells and tissue sections is complete in 60 min, whereas BrdU protocols require harsh denaturation, long blocking steps, and an overnight incubation with anti-BrdU followed by a secondary antibody detection step.

C10337

C10338

C10339

C10340

Click-iT® EdU Alexa Fluor® 488 HCS Assay

Click-iT® EdU Alexa Fluor® 555 HCS Assay

Click-iT® EdU Alexa Fluor® 594 HCS Assay

Click-iT® EdU Alexa Fluor® 647 HCS Assay

495/519

555/565

590/615

650/670

Yes **

 

HCS

Click-iT® EdU Kits for high-content imaging and analysis are available with Alexa Fluor® 488, Alexa Fluor® 555, Alexa Fluor® 594, and Alexa Fluor® 647 azides for detection of the incorporated EdU.

C10350§

C10352§

C10354§

C10356§

Click-iT® EdU Pacific Blue™ FC Assay Kit

Click-iT® EdU Alexa Fluor® 488 FC Assay Kit

Click-iT® EdU Alexa Fluor® 647 FC Assay Kit

410/455

495/515

650/670

Yes **

 

FC

Click-iT® EdU Kits for flow cytometry analysis are available with Pacific Blue™, Alexa Fluor® 488, and Alexa Fluor® 647 azides for detection of the incorporated EdU.

C10418§

C10425§

C10424§

Click-iT® EdU Microplate Assay Kit

568/585

Yes **

 

M

Detect incorporated EdU with Oregon Green® azide, followed by HRP anti–Oregon Green® and Amplex® UltraRed substrate.

C10214

Premo™ FUCCI Cell Cycle Sensor

485/520
555/584

Yes ‡

 

FC, FM, HCS

As a cell progresses through the cell cycle, fluorescence of the Premo™ FUCCI Sensor changes from red to green.

P36232

HCS Mitotic Index Kit

495/519

 

Yes

HCS, FM

Anti–phosphohistone H3 serves as a sensitive index of mitosis and is detected with Alexa Fluor® 488 secondary antibody.

H10293

* Fluorescence excitation and emission maxima in nm, with absorption maximum in parentheses where applicable. † FC = flow cytometry. FM = fluorescence microscopy. HCS = high-content screening and analysis. M = microplate assay. ‡ Fixable probe. After fixation, the GFP and RFP fusions of the Premo™ FUCCI Cell Cycle Sensor can be detected with anti-GFP antibodies and anti-RFP antibodies. § The Click-iT® EdU HCS Assay Kits are available in 2-plate (Cat. No. shown) and 10-plate sizes. The Click-iT® EdU Flow Cytometry Assay Kits are available in a 50-assay (Cat. No. shown) and a 100-assay size. ** In the Click-iT® EdU assay, EdU is incorporated in live cells, but then detected following cell fixation and permeabilization.

 

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