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Cell proliferation reagents measure an expanding population due to cell division
Cell proliferation analysis by flow cytometry is important for drug development and biological processes including (1) measuring compound toxicity, (2) CAR T cell development, (3) inhibition of tumor cell growth during drug development, and (4) diabetes drug development with islet cells. Using flow cytometry, proliferation measurements are typically made based on average DNA content or on cellular metabolism parameters. Assays can report either total or live cell numbers, or measure DNA synthesis in single cells.
Cell Proliferation Assay Protocols Cell Cycle & Proliferation Pathways
We offer dyes, kits, and antibodies to track proliferation. Use our guide to find a reagent for flow cytometry assays or multi-color panels.
Guide for cell proliferation using flow cytometry
Approaches to assess cell proliferation by flow cytometry include: measuring DNA synthesis with Invitrogen Click-iT EdU Flow Cytometry Assay Kits or traditional bromodeoxyuridine (BrdU) staining, tracking generations of cell division with Invitrogen CellTrace Cell Proliferation Kits or carboxyfluorescein succinimidyl ester (CFSE), or alternatively building flow cytometry panels for the detection of proliferation markers such as Ki-67, MCM2, and PCNA.
| Product | Target | Fixable | Multi-plexing | Live-cell analysis | Application |
|---|---|---|---|---|---|
| Click-iT and Click-iT Plus EdU Flow Cytometry Assay Kits | Incorporation into newly synthesized DNA | Yes | Yes | Yes | Cell proliferation |
| BrdU | Incorporation into newly synthesized DNA | Yes | Variable | Yes | Cell proliferation |
| CellTrace Cell Proliferation Kits | Lysine-containing proteins | Yes | Yes | Yes | Generational analysis |
| Ki-67 antibody | Nuclear protein expressed in proliferating cells | Yes | Yes | No | Cell proliferation and cell cycle |
| Minichromosome maintenance (MCM2) antibody | Nuclear protein expressed in proliferating cells | Yes | Yes | No | Cell proliferation and cell cycle |
| Proliferating cell nuclear antigen (PCNA) antibody | Nuclear protein expressed in proliferating cells | Yes | Yes | No | Cell proliferation and cell cycle |
DNA synthesis measurement
A common method to measure DNA synthesis involves the incorporation of a nucleoside analog—typically the thymidine analog BrdU—into newly synthesized DNA in living cells, which can later be detected using a fluorescently labeled antibody that recognizes the analog. The fluorescent signal can be analyzed with flow cytometry instruments to provide a quantitative measurement of DNA synthesis during cell proliferation. Click-iT EdU Flow Cytometry Assay Kits were developed to overcome some of limitations of the BrdU technique.
BrdU is a synthetic nucleoside analog of thymidine. BrdU is incorporated into the newly synthesized DNA and may be targeted by an antibody specific for BrdU.
EdU (5-ethynyl-2’-deoxyuridine) is a thymidine nucleoside analog and is detected with click chemistry. Unlike BrdU, EdU assays do not require DNA denaturation for incorporation. Cells tagged with Click-iT EdU flow cytometry reagent can be measured against total DNA in a cell stained with Hoechst or PI (propidium iodide) or a specific labeled subset.
The Invitrogen Click-iT EdU and Plus EdU flow cytometry assays utilize the power of click chemistry and the modified nucleoside EdU to provide a superior alternative to BrdU staining for detecting and quantitating newly synthesized DNA.
- Accuracy—superior to BrdU assays with minimal variation (low CVs)
- Simplicity—streamlined protocol
- Compatibility—assays allow users to multiplex with GFP, mCherry, APC, PerCP, PE, and other fluorophores (Click-iT Plus EdU kits only)
Figure 1. Dual-parameter plot of human T cells assayed for CD3 and DNA strand breaks. CD3 was detected using an anti-CD3 antibody labeled with PE-Cy7 dye. DNA strand breaks were detected using the Invitrogen Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit.
Dye dilution assays for cell proliferation
CellTrace stains and CFSE are fluorescent tracking dyes that are retained in living cells. With each generation, the amount of dye in each cell is halved, so fluorescence analysis of proliferating populations can be used to quantify the successively decreasing brightness for each generation. These dyes are typically effective at reporting proliferation above background autofluorescence for over six generations. CFSE is a green stain excited at 491 nm and with an emission peak at 521 nm. CellTrace stains are available in 5 fluorescent labels including those excited by the UV, violet, blue, yellow, and far red lasers.
CFSE is a first-generation cell proliferation tracker that covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. When CSFE-labeled cells divide, each progeny cell also contains CFSE. Each generation exhibits a decrease in fluorescence, which can be measured via flow cytometry.
Next-generation CellTrace fluorescent stains are membrane-permeant fluorescent molecules that are retained within the cells through subsequent rounds of cell division, with each daughter cell receiving approximately half of the fluorescence of the parent. CellTrace fluorescent stains can be used without affecting morphology or physiology to trace generations in vivo or in vitro.
- Superior performance—bright, single-peak staining enables visualization of multiple generations
- Long-term signal stability—well-retained in cells for several days post-stain
- Non-cytotoxic—no known effect on proliferative ability or biology of cells
- Compatibility—bright, single-peak staining available in multiple fluorophores
A Novel Ultraviolet Dye for Cell Proliferation Analysis in Flow Cytometry
Figure 2. Labeling of peripheral blood mononuclear cells (PBMCs) to study multiple cell generations. PBMCs were stained with (A) 5 μM CellTrace Violet, (B) 2 μM CellTrace CFSE, (C) 10 μM CellTrace Yellow, and (D) 2 μM CellTrace Far Red reagents. Dynabeads Human T-Activator coupled with CD3/CD28 antibodies were used for T cell expansion and activation. Samples were incubated in OpTmizer T-Cell Expansion Medium at 37°C/5% CO2 for 7 days. Samples were analyzed using SYTOX Green or SYTOX Red dead cell stains to gate on live cells and mouse anti-human CD4 Pacific Blue or CD4 FITC were used to gate on proliferating cells. The gray peaks represent unstimulated control cells (parent generation) and the peaks to the left of each gray peak represent individual generations of cells that proliferated during the course of the experiment. (E) PBMCs were stained with 10 μM CellTrace Blue reagent, stimulated, and kept in culture for 5 days. The gray peak represents the unstimulated control cells. The discrete blue peaks represent individual generations of cells that proliferated during the course of the experiment.
Cell cycle phases coordinate multiple proteins to synthesize DNA and divide cells. The Invitrogen portfolio of antibodies for flow cytometry provides:
- Choice—13,000 antibodies for flow cytometry, including Invitrogen eBioscience conjugates
- Variety—build your panels with a wide variety of fluorescent conjugates to build large multi-color panels
- Trusted source—validated antibodies for quality results
Figure 3. Intracellular staining using Ki-67 antibody followed by flow cytometry analysis. Intracellular staining of mouse splenocytes unstimulated (left) or stimulated for 2 days with immobilized Anti-Mouse CD3 Functional Grade (right) with Anti-Human/Mouse CD45R (B220) PE and 0.03 μg of Anti-Mouse/Rat Ki-67 APC using the Foxp3/Transcription Factor Buffer Set and protocol. Total viable cells, as determined by Fixable Viability Dye eFluor 450, were used for analysis.
Figure 4. Examine the expression of Ki-67, proliferating cell nuclear antigen (PCNA), or minichromosome maintenance protein 2 (MCM) in your proliferating sample. These proteins can be found in the cell cycle.
Assaying cell proliferation with imaging
Cell proliferation can be quantitated and spatially detected with microscopy and other high-content imaging systems. A broad range of assays including the Click-iT EdU assays for imaging and Invitrogen CyQUANT Cell Proliferation Assays for microplate readers.
Figure 5. Click-iT EdU was incorporated into supporting cell nuclei.
Immunology at work
Learn immune cell type markers and protocols. The Invitrogen Immunology at Work Resource Center is a learning center with technical content designed for new and experienced life scientists alike exploring the field of immunology.
Resources
Fluorophore and reagent selection guide for flow cytometry
Download Flow Cytometry Protocols Handbook
Spectral Flow Cytometry Fundamentals
Invitrogen eBioscience Resources—Selection guides, Best Protocols, product performance and more.
Intracellular Staining for Flow Cytometry How-To Video—for detecting cytokines and intranuclear markers.
Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.
5 Steps Resources
Support
Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.
Flow Cytometry Panel Design Support—Work with one of our technical sales specialists to discuss your experimental needs and guide you through the process.