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Cell Proliferation Assays

Cell proliferation assays are valuable tools with a wide range of applications in cell biology and drug-discovery research. They are commonly used to assess normal cell heath and are essential for evaluating the antiproliferative potency and compound toxicity of novel chemotherapeutic agents. AAT Bioquest offers several strategies for assessing cell proliferation based on tracking generations of cell division or monitoring critical parameters of cell health, including metabolic activity, DNA synthesis, or ATP concentration. Select from a broad range of traditional colorimetric reagents for determining cell proliferation, in particular tetrazolium salts, and several highly sensitive fluorescent-based assays for flow cytometry, imaging, and microplate applications.

 



Selecting the Right Cell Proliferation Assay


The most accurate method for assaying cell proliferation is to measure newly synthesized DNA via BrdU (5-bromo-2'-deoxyuridine) incorporation during DNA replication. Although highly accurate and reproducible, this method can be rather tedious as subsequent detection with fluorescent anti-BrdU antibodies is necessary to measure proliferation. Alternatively, cell proliferation can be assessed more rapidly by measuring the rate of metabolic activity with WST, resazurin, or cellular esterase cleaved dyes or tracking generations of cell division with CytoTell™ dyes or CFSE. Ultimately, selecting the cell proliferation assay best suited for your purpose depends mainly upon cell type, research protocol, and the action mechanism you wish to study. The following table provides an overview of the types of cell proliferation assays available.

 

Table 1. Methods for measuring cell proliferation

Action Mechanism
Assay and reagents to use
Platform
Principle
Newly synthesized DNA
  • Bucculite™ XdU assays
  • Bucculite™ FdU assays
  • BrdU
  • Flow cytometry
  • Fluorescence microscopy
  • Microplate reader
During the S phase, thymidine analogs are incorporated into newly synthesized DNA and then detected using fluorescent antibody probes.
Metabolic Indicators
  • WST and MTT (colorimetric output)
  • Resazurin (fluorimetric output)
  • Calcein AM dyes (fluorimetric output)
  • Microplate reader
Indirectly detects proliferation by monitoring metabolic activity. Healthy cells reduce substrates which can be read by absorbance or fluorescence.
Tracking generations of cell division
  • CytoTell™ dyes
  • CFSE
  • Flow cytometry
Labels live cells with fluorescent tracking dyes. As cells divide, the fluorescent dye is uniformly distributed between daughter cells, resulting in the progressive halving of fluorescence intensity with each successive generation.
ATP concentration
  • PhosphoWorks™ ATP assays
  • ReadiUse™ ATP assays
  • Microplate reader
Dead or dying cells contain little to no ATP, creating a strick linear relationship between the concentration of ATP measured in a cell lysate and the number of cells. Therefore, ATP determination assays can be used to assess proliferation.

 

DNA Synthesis Cell Proliferation Assays


Measuring newly synthesized DNA in situ is the most reliable and precise method for identifying proliferating cells. One way to accomplish this involves incorporating the thymidine analog, BrdU (17030), into newly replicated DNA during the S phase of the cell cycle. Proliferating BrdU-labeled cells can then be measured using fluorescently labeled anti-BrdU antibodies. BrdU staining is suitable for various applications, including IHC, ICC, and ELISA, and is amenable to staining with other biomarkers of interest in flow cytometry. We offer fluorescent conjugates of the mouse monoclonal anti-BrdU antibody clone Bu20a labeled with PE (V103305) and unlabeled mouse monoclonal anti-BrdU antibody clone Bu20a (V103300) and clone MoBu-1 (V103295) that can be labeled with our brightest and most photostable iFluor™ dyes.

Click-Chemistry Driven Bucculite™ XdU Cell Proliferation Assays


Unlike BrdU assays, Bucculite™ XdU Cell Proliferation Assays do not rely on antibodies to quantify nascent DNA, nor do they require DNA denaturation needed to facilitate antibody detection of BrdU-labeled nucleosides. Rather, Bucculite™ XdU Cell Proliferation Assays use a proprietary mixture of alkyne-containing thymidine analogs, XdU, that is incorporated into newly synthesized DNA and subsequently detected via a copper-catalyzed azide-alkyne cycloaddition (i.e., click reaction, Figure 1).


Bucculite™ XdU Cell Proliferation Assay Principle. Proliferating cells in the presence of XdU incorporate the compound at thymidine bases during the S phase. Fluorophore-labeled azide reacts with the incorporated XdU to allow detection by imaging or flow cytometry (figure made in BioRender).


Since the click reaction utilizes biorthogonal moieties to detect proliferating cells, background interference is minimal. More importantly, because of the mild reaction conditions, Bucculite™ XdU Cell Proliferation Assays preserve cell morphology, antigen-binding sites, and sample integrity, affording the user an opportunity for multiplexing analysis with cell cycle dyes, antibodies against cell surface markers, and fluorescent proteins. After XdU incorporation, the click reaction and subsequent wash steps can be completed in less than 60 minutes, and nascent DNA can be quantified using standard imaging systems or a flow-cytometer. Also available are environmentally safe copper-free Bucculite™ FdU Cell Proliferation Assays in green, red, and deep red fluorescence. These kits utilize our patent Buccutite™ labeling chemistry to facilitate the detection of newly synthesized DNA in actively proliferating cells.


Cell proliferation was detected using the Bucculite™ XdU Cell Proliferation Assay. HeLa cells were processed using the reagents and fixation/detection protocol provided in the Bucculite™ XdU Cell Proliferation iFluor™ 488 Imaging kit (22326) and the Bucculite™ XdU Cell Proliferation iFluor™ 647 Imaging kit (22328). Both samples were counter-stained with Hoechst® 33342 nucleic acid stain (17530) and imaged using a fluorescence microscope.


 

Table 2. Bucculite™ Cell Proliferation Assays for flow cytometry and imaging.

Product
Unit Size
Cat No.
Bucculite™ Flow Cytometric XdU Cell Proliferation Assay Kit *Blue Laser-Compatible*100 Tests22323
Bucculite™ Flow Cytometric XdU Cell Proliferation Assay Kit *Red Laser-Compatible*100 Tests22325
Bucculite™ XdU Cell Proliferation Fluorescence Imaging Kit *Green Fluorescence*200 Tests22326
Bucculite™ XdU Cell Proliferation Fluorescence Imaging Kit *Red Fluorescence*200 Tests22327
Bucculite™ XdU Cell Proliferation Fluorescence Imaging Kit *Deep Red Fluorescence*200 Tests22328
Bucculite™ FdU Cu-Free Cell Proliferation Fluorescence Imaging Kit *Green Fluorescence*200 Tests22305
Bucculite™ FdU Cu-Free Cell Proliferation Fluorescence Imaging Kit *Red Fluorescence*200 Tests22315
Bucculite™ FdU Cu-Free Cell Proliferation Fluorescence Imaging Kit *Deep Red Fluorescence*200 Tests22320
BrdU [5-Bromo-2'-deoxyuridine] *CAS 59-14-3*25 mg17030
BrdUTP [5-Bromo-2'-deoxyuridine 5'-triphosphate] *10 mM in TE Buffer* *CAS 102212-99-7*100 ul17031

 

Dye Dilution Assays for Cell Proliferation


CFSE and CytoTell™ indicators are fluorescent tracking dyes useful for monitoring cell proliferation in living cells by flow cytometry. As cells divide, the fluorescent dye is uniformly distributed between daughter cells, resulting in the progressive halving of fluorescence intensity with each successive generation. These proliferation dyes can be used to track cell proliferation long-term both in vitro and in vivo for several generations.

Proliferation Assay Using CFSE


CFSE (Carboxyfluorescein diacetate, succinimidyl ester, 22022) and ReadiUse™ CFSE (22028) are cell-permeable green fluorescent proliferation indicators that emit a fluorescence signal at 517 nm when excited by the 488 nm argon-ion laser. Both dyes passively diffuse across uncompromised cell membranes, whereby enzyme reactions with intracellular esterases cleave acetate groups from CFSE to yield an amine-reactive carboxyfluorescein, SE that covalently binds to proteins within the cell. While CFSE remains widely used as a green fluorescent proliferation indicator excited at 498 nm, it is, however, not without caveats. CFSE is highly toxic to cells, susceptible to dye leakage, and is known to spill over into the PE and PE-Texas Red channels.

CytoTell™ Proliferation Indicators


CytoTell™ dyes are a series of cell-permeable fluorescent tracers designed to monitor cell proliferation in living cells without any of the drawbacks of CFSE. Upon passive diffusion across cell membranes, CytoTell™ dyes are hydrolyzed by intracellular esterases to yield highly fluorescent amine-reactive or thiol-reactive dyes that irreversibly bind to amine or thiol groups on intracellular proteins. Cytotoxicity is minimal, and the dyes are well-retained within cells through several generations of division. Visualize up to 9 generations using CytoTell™ UltraGreen (22240). CytoTell™ dyes are available in 7 different fluorescence emission colors offering greater flexibility in multicolor analysis. They may be fixed and permeabilized to analyze intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers.


Cell proliferation assay using CytoTell™ Green and CFSE. Jurkat cells (~2x106 cells/mL) were stained with CytoTell™ Green or CFSE on Day 0. Cells were then passed serially at a 1:1 ratio on the day specified. Fluorescence intensity was measured with ACEA NovoCyte 3000 flow cytometer in the FITC channel on the day of passage. Different color peaks represent successive generations.


 

Table 3. CytoTell™ and CFSE cell proliferation dyes for flow cytomtery

Product Name
Excitation Source (nm)
Ex (nm)
Em (nm)
Unit Size
Cat No.
CytoTell™ BlueViolet Laser (405 nm)410445500 Tests22251
CytoTell™ Violet 500Violet Laser (405 nm)415499500 Tests22248
CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]Blue Laser (488 nm)49851725 mg22022
ReadiUse™ CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]Blue Laser (488 nm)4985175 x 500 µg22028
CytoTell™ GreenBlue Laser (488 nm)510525500 Tests22253
CytoTell™ UltraGreenBlue Laser (488 nm)492519500 Tests22240
CytoTell™ OrangeGreen Laser (531 nm)541560500 Tests22257
CytoTell™ Red 590Green Laser (531 nm)560574500 Tests22261
CytoTell™ Red 650Red Laser (633/647 nm)626643500 Tests22255
CytoTell™ BlueViolet Laser (405 nm)4104452 x 500 Tests22252

 

Metabolic Assays for Cell Proliferation


Metabolic indicators, such as tetrazolium salts and resazurin, measure the cellular reduction potential in metabolically active cells to evaluate proliferation. Following reduction, these substrates yield bright colorimetric products that can be measured using a spectrophotometer or microplate reader in low- or high-throughput configurations. Reduced resazurin can also be measured using a fluorescence microplate reader.

Proliferation Assay Using Tetrazolium Salts



Cell proliferation was determined using Cell Meter™ Colorimetric MTT Cell Proliferation Kit. HeLa cells at 0 to 40,000 cells/well/100 µL were added to a clear bottom 96-well plate overnight. The absorbance was measured at 560 nm using a SpectraMax reader (Molecular Devices). 500 cells/well was detected compare to ~5,000 cells/well with Sigma's MTT assay kit.
The four types of tetrazolium salts used to measure proliferation are MTT, MTS, XTT, and WST-8. Reduction of MTT is the most common, however, for most commercial MTT assays, solubilization of the purple formazan product in DMSO or isopropanol is required before quantification. The convenient Cell Meter™ Colorimetric MTT Cell Proliferation Kit (22768) simplifies the task of counting cells to an easy mix and read format without the need for solubilization. The concentration of formazan determined by optical density at 560 nm is directly proportional to the number of live cells.
 

Cell proliferation was determined using Cell Meter™ Colorimetric WST-8 Cell Quantification Kit. HeLa cells at 0 to 10,000 cells/well/100 µL were added in a clear-bottom 96-well plate. The absorbance was measured at 460 nm using a SpectraMax reader (Molecular Devices).
Among the other tetrazolium salts, the water-soluble WST-8 offers the highest sensitivity. Reduction of WST-8 by cellular dehydrogenases yields water-soluble orange formazan crystals that do not require solubilization prior to quantification. The concentration of formazan product can be determined by measuring absorbance at 460 nm and is proportional to the number of living cells. The Cell Meter™ Colorimetric WST-8 Cell Quantification Kit provides a sensitive assay with exceptional linearity of up to ~106 cells per well. No pre-mixing of components is required, as the WST-8 substrate is conveniently provided in a solution that can be directly added to the cells. Each kit provides sufficient reagents for either ~1000 (22770) or ~5000 tests (22771) using a standard 96-well microplate. WST-8 is also available as a stand-alone reagent in 25 mg (15705), 100 mg (15706), and 1 g (15707) unit sizes supplied as 50 mM aqueous solutions.

 

Table 4. Colorimetric MTT and WST-8 tetrazolium salt-based assays for measuring cell proliferation.

Product
Unit Size
Cat No.
Cell Meter™ Colorimetric MTT Cell Proliferation Kit1000 Tests22768
Cell Meter™ Colorimetric MTT Cell Proliferation Kit5000 Tests22769
Cell Meter™ Colorimetric WST-8 Cell Quantification Kit1000 Tests22770
Cell Meter™ Colorimetric WST-8 Cell Quantification Kit5000 Tests22771
ReadiUse™ WST-8 *50 mM aqueous solution*25 mg15705
ReadiUse™ WST-8 *50 mM aqueous solution*100 mg15706
ReadiUse™ WST-8 *50 mM aqueous solution*1 g15707


Proliferation Assay Using Resazurin


Like the aforementioned tetrazolium salts, the oxidation-reduction indicator resazurin (15700) can be useful for quantifying cell proliferation. Following reduction, the blue non-fluorescent resazurin yields a pink-colored and highly fluorescent resorufin product that is compatible with both fluorescence and absorbance microplate readers. The fluorescent or colorimetric signal is proportional to the number of living cells in the sample. Resazurin has been reported to be successfully used in a wide range of cell types, including bacteria, yeast, fungi, protozoa, and cultured mammalian cells.

 

Table 5. Product specifications for resazurin.

Product
Abs (nm)
Ex/Em (nm)
Unit Size
Cat No.
Resazurin, sodium salt *CAS 62758-13-8*605571/584100 mg15700

 

ATP Assays for Cell Proliferation



Jurkat cells were measured using the PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free* on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect as low as 100 cells. The integration time was 1 second.
ATP, which is present in all metabolically active cells and virtually non-existent in dead cells, is a suitable biomarker for identifying viable cells. Assays for quantifying ATP have been used to determine cell proliferation and cytotoxicity in both bacterial and mammalian cells and for detecting low-level bacterial contamination in samples such as water, soil, and blood. While several detection methods can be used, researchers commonly choose luciferin-luciferase bioluminescence-based ATP assays to measure cell proliferation due to its higher sensitivity. The PhosphoWorks™ Luminometric ATP Assay Kits provide a simple and homogeneous method for measuring ATP using firefly luciferase and its substrate luciferin. In the presence of Mg2+, firefly luciferase catalyzes the reaction of luciferin, ATP, and O2 to produce light at ~560 nm that can be detected using a luminometer. The concentration of ATP present is proportional to the number of viable cells in the sample. Also available is a PhosphoWorks™ Colorimetric ATP Assay Kit (21617) and a PhosphoWorks™ Fluorimetric ATP Assay Kit (21620) that can detect ~3 µM or ~0.4 µM of ATP in a 100 µL reaction volume, respectively.

 

Table 6. ATP assays for measuring cell proliferation.

Product
Unit Size
Cat No.
PhosphoWorks™ Colorimetric ATP Assay Kit100 Tests21617
PhosphoWorks™ Fluorimetric ATP Assay Kit100 Tests21620
PhosphoWorks™ Luminometric ATP Assay Kit *Steady Glow*1 Plate21609
PhosphoWorks™ Luminometric ATP Assay Kit *Steady Glow*10 Plate21608
PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*1 Plate21610
PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*10 Plate21621
PhosphoWorks™ Luminometric ATP Assay Kit *DTT-Free*1 Plate21612
PhosphoWorks™ Luminometric ATP Assay Kit *DTT-Free*10 Plate21613
Readiuse™ Rapid Luminometric ATP Assay Kit100 Tests21601
Readiuse™ Rapid Luminometric ATP Assay Kit1000 Tests21602