The monitoring of cell proliferation is central to the evaluation of the physiological state of cells and their responses to drugs, environmental factors, and the progression of cellular disease states. Cell proliferation analysis is used to support many areas of clinical investigation including immunophenotyping, drug effects and toxicity, as well as T cell behavior under various conditions. Assays that measure cell proliferation in individual cells work by monitoring changes in intracellular markers and/or DNA.
Flow cytometry is an ideal platform for measuring cell proliferation and enables us to study various unique cell types within a heterogeneous population. The most common approaches to monitoring cell proliferation by flow cytometry involve the use of fluorescent dyes such as carboxyfluorescein diacetate succinimidyl ester (CFSE) and PKH26 that incorporate into the cell cytosol, cell membranes and intracellular structures of living cells. A decrease in fluorescence occurs as dyes are evenly diluted into daughter cells with subsequent cell divisions representing the successive generations, and these are identified as distinct peaks during flow cytometry analysis (Fig. 1).
Cell proliferation dyes can be used to track cell divisions in vivo or in vitro and many of these staining procedures can withstand fixation and permeabilization and are compatible with subsequent immunostaining. Advances in cytometer technology and integrated software have improved data collection and decreased hands-on time, but more importantly, enable better quality and more in-depth information to be gathered for each sample. In this blog, we review the different types of staining dyes and advanced techniques that are now available, and the experimental designs that can be implemented for their optimum use.
Fig. 1. The principal of dye dilution assays for Cell Proliferation. Parental cells are labeled with tracking dye on day 0. Flow Cytometry analysis can reveal successively dimmer peaks representing each generation of cells from that parental generation.
Nucleotide Incorporation Dyes:
Bromodeoxyuridine (BrdU) incorporation has long been used to assess DNA synthesis in vivo and in vitro. The foundation of this method is the incorporation of BrdU as a thymidine analog into the nuclear DNA during the S-phase of the cell cycle, that can be tracked using antibody probes (Fig. 2). It is much safer and easier to use than older systems such as 3H-thymidine incorporation, and although considered toxic, the concentration that BrdU is used for labeling is typically not an issue.
There are several key points to consider when designing a BrdU labelling experiment:
- It is important to make sure that the reagent BrdU is freshly prepared for each experiment since this reagent has a short half-life under working conditions.
- Denaturation (unwinding) of the DNA is a critical step in the process- too little denaturation and the BrdU will not integrate, too much and the DNA can be damaged or degraded. It is therefore important to use a validation protocol for this step. If acidic conditions are used to denature the DNA, then make sure the sample is adequately washed post labeling to ensure acidic denaturation of the detection antibody is avoided. Coupling with conjugated monoclonal antibody labeling can be helpful to identify cellular subsets, however it is important to avoid protein fluorochromes such as PE and APC that are not conducive with the processing.
- Data interpretation can be challenging. Nucleotide incorporation only reflects the cellular population in S-phase, a very short phase in cellular division. This population progresses through G2 and then into G1- so make sure this is considered when reviewing and interpreting data.
Fig. 2. Jurkat cells were pulsed with 10µM BrdU for 1 hours, using the acid denaturation method and stained with anti-BrdU-APC. Cells were stained with 7-AAD for DNA analysis.
Recently, new variations on the BrdU assay technique have been commercialized including the (5-ethynyl-2’-deoxyuridine) EdU assay that uses Cu-induced Click-Chemistry to provide a means of labeling DNA without the need for secondary antibody detection, and with reduced cellular toxicity. Four different fluorescent probes, with excitations of 488nm, 594nm and 647nm enable multiplexed flow cytometry panels to be designed to assess cell proliferation in complex heterogeneous cell populations.
Protein Binding Dyes
Protein binding dyes such as succinimidyl ester family of dyes that includes Carboxyfluorescein diacetate, succinimidyl ester (CFSE), CellTrace Violet, and eFluor Proliferation dye 670, have been used for years to track cell division. Although CFSE remains a popular choice, there are some drawbacks to its use including the potential of cell toxicity, leakage from labelled cells as well as bleed-through of the signal into the PE channel. As a result, derivatives of CFSE are now commercially available that mitigate some or all of these issues.
There are important nuances to the optimal use of these protein binding dyes that include many principles for good flow cytometry techniques. Uniform labeling of cell cytosol is the foundation of good cell proliferation analysis using these methods These factors include:
- The avoidance of proteins in the labelling buffer,
- Ensuring adequate washing and incubation to remove unbound label prior to analysis.
- As with most flow cytometry reagents, titration of the dye on the cell population of interest is really important for clean data. Different cell types will stain to saturation at different levels of protein dye, so titrate on the population of interest whenever possible.
- The utilization of brightest protein staining dyes enables highest numbers of cell division to be tracked, so plan your experiment accordingly- labeling intensity is very important.
- If looking for cell proliferation, always include an unstimulated control population.
- It is always good practice to exclude any dead cells from the analysis using viability dyes.
Membrane-binding dyes, typified by PKH26 are highly lipophilic dyes that partition stably and non-covalently into the membranes of cells. Lipophilic tracking dyes can be used on almost any cell type, to study a wide range of functions from immune cell function to migration, proliferation, antigen presentation and cellular adhesion.
There are some critical steps for the successful use of lipophilic dyes for cell proliferation studies. These include:
- the rapid mixing of the staining dye with the cells to ensure uniform cell staining.
- the careful selection of diluent used in this step. This diluent is typically salt-free and low in protein diluent. This achieves several goals: minimizes cell toxicity by maintaining the osmotic pressure of the mixture and maximizes dye solubility during the rapid mixing of the dye with the cells that ensures high staining efficiency with optimal dye dispersal throughout the sample.
- the final staining intensity is driven by both the cell and dye concentrations, and preliminary titration of dye with the target cell population of interest is always recommended.
- control of the length of staining. Since the labelling of the cells takes place by partitioning rather than equilibrium binding, the uptake of the dye into the membrane continues for as long as the cells and dye are in contact. If left for too long, the membranes of the cells can become compromised.
- the staining process can be halted by the addition of buffered salt solutions or culture media to act as reservoirs for any unbound dye, however this may not eliminate dye transfer to unlabeled cells, and therefore, a solution of FBS or 10-15mg/mL albumin may also be employed as a stop solution.
There are several alternative unique staining compounds that are typically used for cell viability but can also be applied for cell proliferation assessment.
Resazurin (labels live cells)
Resazurin is a fluorescent dye for the quantitative measurement of cell proliferation, viability, and cytotoxicity. This is a non-toxic, water soluble, redox-sensitive dye that changes from blue to pink (high fluorescence) upon reduction Read at 544 Excitation, 590nm emission. This dye enables the metabolic activity of cells to be readily assessed.
Calcein AM (live cells)
Calcein AM is a non-fluorescent method to accurately measure cell viability and/or cytotoxicity on a wide variety of cell types and states: proliferating or non-proliferating cells, suspension or adherent, rapid, HTS-compatible. Calcein AM is a non-fluorescent, hydrophobic compound that easily permeates intact live cells. It is hydrolyzed by intracellular esterases to produce calcein, a hydrophilic strongly fluorescent compound that is retained in the cell cytoplasm. Highly applicable for studies including cell adhesion, chemotaxis, MDR, cell viability, apoptosis and cytotoxicity.
MTT Cell Proliferation/Viability Assay (live cells)
MTT can be used to safely assess cell proliferation, cell viability, and/or cytotoxicity. MTT is reduced to an insoluble formazan dye by mitochondrial enzymes associated with metabolic activity, through NADH and NADPH.
TACS XTT Cell Proliferation/Viability Assay (live cells)
XTT can be used to assay cell proliferation, cell viability, and/or cytotoxicity. The procedure gives reproducible and sensitive results; however, it has been shown to overestimate viable cell counts.
Propidium iodide (dead cells)
Red-fluorescent DNA stain; membrane impermeant to live cells.
DAPI (live cells)
A standard fluorescent DNA stain that binds to AT-rich regions and can permeabilize cells at high concentrations.
Hoechst 33342 (total cells)
Chromatin-enhanced Hoechst 33342 fluorometric assay represents a simple, reproducible, and rapid way of analyzing cell proliferation. It is recommended that cells are stained with Hoechst just before acquisition to minimize the decline of fluorescence over time, and since Hoechst staining intensity is influenced by cell number, a standardized staining protocol should be employed.
Intracellular Markers of Cellular Proliferation
The proliferative activity of cells can also be measured by the immuno-detection of cell cycle-specific antigens such as Ki-67, mini-chromosome maintenance 2 (MCM2) or proliferating cell nuclear antigen (PCNA). All three of these biomarkers are expressed during cell division of both normal and neoplastic cells. Ki-67 represents a non-histone nuclear biomarker that is expressed throughout the active phase of the cells cycle, with the exception of G0 and early G1. The staining index for this marker has recently become a predictive and prognostic biomarker for malignancies including breast cancer (Esteva and Hortobagyi, 2004; Wang et. al. 2016). With Ki-67 representing such an important clinical marker, several studies have focused on the development and validation of robust staining protocols for flow cytometry analysis. Key points for consideration include:
- sample collection process and type (e.g. fresh blood versus CytoChex etc.) and the stability of the sample over time,
- the selection of permeabilization buffer with the least impact on surface staining,
- Ki-67 clone selection.
Fig. 3 Analysis of Cell Proliferation Using a Combination of Cell Trace Violet and Ki-67 expression profiling.
PBMCs were stimulated with 1:2 ratio of CD3/CD28 Dynabeads (Beads:Cells) for 3 days in 10% FBS RPMI 1640 complete media + P/S with 0.1µg/mL CD3 and 30IU IL-2. Fig. A. PBMCs unstimulated (left) and Stimulated (right), gated on CD3+ T-cells. With stimulated cells undergoing multiple generations of proliferation as shown by the Cell Trace Violet Staining and confirmed with Ki-67 staining, compared with unstimulated cells. Fig. B. PBMCs gated on CD3+ T-cells, overlay of unstimulated (shaded peak) and stimulated (blue line, clear peak) cells.
Whatever staining process you utilize, there are other experimental considerations that will ensure the optimal assessment of proliferation using flow cytometry.
- Instrument Selection: using flow cytometers equipped with multiple lasers and detectors is always preferred for efficient and clean multiparameter analysis.
- The inclusion of absolute cell counts can enhance cell proliferation analysis by providing a reliable readout of cell number (and therefore cell division) over the course of the experiment.
- Time points selection for data analysis. Careful consideration of the appropriate time points for cell collection will help in the detection and resolution of as many cellular generations as possible. T-cell activation assays may involve several days of stimulation before proliferative assessment, whereas PBMC-assays may require time point collection ranges of 4-5 days or longer. Too little proliferation, and you may not see any cell divisions, whereas too much proliferation will decrease the resolution of the division peaks. It is always recommended to determine a saturating concentration by running a titration curve using halving dilutions of activating antibody or compound, and always include an unstimulated control that does not have any activating antibody/compound to determine the amount of CFSE in generation 0 cells that have not divided.
- Optimizing the dye retention and antibody titration for the target cell population of interest enables more precise information on the proliferation state of that cell population. Although higher dye concentrations result in brighter cells and the ability to observe more divisions, these concentrations may also impact cellular function and viability. To account for this, it is typical to start with a labeling solution between 1-10 µM for 1-20 million cells/mL and optimize incubation time, temperature, and efficient and effective removal of excess dye. For example, a typical CFSE stain with PBMCs utilizes a 5µM stain in a solution containing 5% FBS or BSA, for 5 minutes at room temperature before rinsing. Each defined peak represents one generation, and typically 6-8 peaks are optimal for thorough analysis. In order to achieve this, it is important to ensure that the dye is not causing cell death either immediately or after long term culture.
- For proliferative assessment using flow cytometry, it is important that an unlabeled control is used to exclude non-specific binding, and cellular autofluorescence, and employ the same PMT voltages and compensation matrix across samples.
- Given that proliferation assays are typically limited by sample run time, having an automated plate loader on your flow cytometer can markedly increase efficiency.
The Nuances of Analyzing Cell Proliferation Data
Software solutions are available to analyze readouts for cellular proliferation assays. FlowJo for example offers a Proliferation Platform that can be used to calculate the following indices.
Division index is the average number of cell divisions that a cell in the original cell population has gone through, including those that have not undergone division.
Proliferation index is calculated from the total number of divisions divided by the number of cells that went into the division. Since it only considers the cells that underwent at least one cell division it gives a more comprehensive understanding of how fast the responding cells are growing.
Expansion Index determines the fold-expansion of the overall culture. For example, if 5 million cells are stimulated, and the EI is determined to be 2.0, then the resulting cell count at the end of the experiment is 10 million cells.
Replication Index determines the fold expansion of only the responding cells.
Including the division and proliferation indexes provides more information on how well cells are growing within a population. The division index tells you the average number of divisions of each cell revealing how proliferative cells are in comparison to each other and gives a good idea of cell proliferation of all cells. The proliferation index indicates the number of divisions completed on average per cell that is proliferating and can reflect the kinetics of cell behavior under different conditions and samples.
The success of flow cytometry-based cell proliferation assays relies on the careful selection of probes and experimental design. Taking time to establish appropriate timepoints for cell collection and analysis will ensure you are able to detect a higher number of divisions as possible. By titrating dyes and antibodies on the target cell population, you will be able to obtain more precise information on the proliferation state of your target cells.
Experiments using PBMCs should plan for timepoint collection ranges of 4-5 days, with optimized dye concentrations to limit cell toxicity and surface marker staining to include phenotypic characterization.
At FlowMetric we incorporate robust controls into our experimental design, including unstained samples, unstimulated controls and absolute cell count beads, to help ensure the quality and robust performance of our cell proliferation assays, coupled with fit-for purpose analytics that best address your research needs.
A sincere thank you to Sarah Casanova for generating data in support of this blog.
Authored by: Dr. Julie Bick
Dr. Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Torrey Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.
Esteva FJ, Hortobagyi GN. Prognostic molecular markers in early breast cancer. Breast Cancer Res. 2004;6:109
Wang J, Sang D, Xu B, et al. Value of breast cancer molecular subtypes and Ki67. Expression for the prediction of efficacy and neoadjuvant chemotherapy in a Chinese population. Medicine (Baltimore) 2016;95:e3518.