This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.
Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.
The tumor suppressor gene, p53, is an essential regulator of a number of cellular processes, including cell cycle arrest, apoptosis and senescence1. It is responsible for maintaining genomic stability, and is therefore crucial for maintaining the balance of cell death and cell growth. Mutations in p53 are common in cancer and are the major cause of p53 inactivation leading to uncontrolled cancer cell proliferation2. Interestingly, mutations in p53 only account for approximately 25% of breast cancers3, suggesting that other mechanisms are responsible for the loss of p53 function. The recently discovered p53 isoforms have been shown to be overexpressed in a number of human cancers, and can modulate p53 function4,5. We have previously shown that the p53 isoform, Δ40p53, is the most highly expressed isoform in breast cancer, and is significantly upregulated in breast cancer cells, when compared to normal adjacent tissue6. Following this, we stably transduced the human breast cancer cell line MCF-7 to overexpress Δ40p53 using the LeGO-iG2-puro+ vector (GFP+)7. These cells were used to investigate if high Δ40p53 expression increases cell proliferation rates in breast cancer cells.
There are many direct and indirect methods of measuring cell proliferation of cultured cells in vitro8,9. These can be performed either as continuous measurements over time, or as endpoint assays10. Conventional methods are still useful, such as cell counting using a hemocytometer. This assay is a low cost and direct measure of the cell number, but it does rely on large cell counts and highly skilled training to minimize error and large standard deviations from the counts. The need to perform measurements compatible with high-throughput formats has led to the development of multiwell-plate assays. These luminescence-based assays measure cell numbers based on a luminescent signal that is proportional to the metabolic activity of the cell11,12. More recently, the introduction of high content imaging platforms has allowed for new tools which monitor cell proliferation while providing quantitative and qualitative phenotypic data collection, and includes a variety of systems13. All of these methods provide avenues to measure cell growth, either by continuous measurement or endpoint assays, and each possess a range of advantages and disadvantages with regards to sensitivity, throughput of sample numbers, and cell information, all of which can be weighed accordingly depending on the research question.
This protocol describes three different methods for measuring cell proliferation in vitro, with each method utilizing different ranges of sensitivity, reproducibility and multi-well plate formats. This protocol aimed to compare the use of a hemocytometer counting chamber, a luminescence-based cell viability assay, and cell imager, in the measurement of cell proliferation over a 96 hour time course. To do this, the growth of vector-transduced cells (MCF-7-LeGO) was compared to cells transduced to overexpress Δ40p53 (MCF-7-Δ40p53), using three different cell densities. Cell proliferation was measured every 24 hr for up to 96 hr. Each method was found to have its own advantages and disadvantages, and depending on the aim of the experiment, each still is a valuable method for providing information on the rate of proliferation.
1. Preparing Cells for Proliferation Assays
Note: Prepare the two cell lines in the same manner and seed in the same format for each method to be analyzed.
2. Determining Cell Count Using a Hemocytometer
3. Determining Cell Proliferation Using a Luminescence-based Assay
Note: This is an endpoint measurement. Once the reagent is added to the cells, the plate can only be quantified once.
4. Determining Cell Count Using a Cell Imager
To study different methods of measuring the proliferation of cultured cells, the cell proliferation of MCF-7-Δ40p53 transduced cells was compared to the non-transduced MCF-7-LeGO breast cancer cell line. The three methods that were compared – the conventional hemocytometer method, cell viability luminescence assay, and cell imaging analysis- are outlined in the schematic diagram (Figure 1). Each method has advantages and disadvantages to accurately measure cell counting over time, and the most effective method depends on the endpoint requirements of the experiment and the information that can be acquired for a cell population.
The growth of MCF-7-LeGO and MCF-7- Δ40p53 cells were measured after 24 hr for 4 days. As shown in Figure 1, the two cell lines were seeded at three different cell densities (1.5 x 103; 3 x 103; 5 x 103 cells/well) and cell counting was performed 24, 48, 72 and 96 hr after seeding. Each method demonstrated increased cell proliferation, using a hemocytometer, a luminescence-based assay, and a cell imager (Figures 2a, b and c, respectively) over 96 hr. The greatest increase in cell proliferation was seen at the highest cell density (5 x 103 cells), and also showed the most reproducible results at each time point, suggesting this to be the optimum cell density for measuring proliferation in these cells. There was no significant difference in cell proliferation between the cell lines.
The measurement of cell proliferation between the different methods was compared by a linear regression analysis6 (Figure 3; Table 4). There was a significant correlation between each of the different methods tested, when comparing cell proliferation at all of the cell densities in both cell lines (Figure 3a and b). The strongest correlation was observed between the comparison of the luminescence-based assay, and the cell imager (R2 = 0.8899, p ≤0.0001; R2 = 0.9805, p ≤0.0001, Figure 3a and b, respectively).
A visual representation of cell proliferation using the cell imager is shown (Figure 4). As this method uses continual measurements of a single plate, the cells can be imaged every 24 hr until the cells reach close to 100% confluence. As shown in Figure 2, the growth rates of the MCF-7-LeGO and MCF-7-Δ40p53 cells were comparable across all time points. This method provides useful cellular information, including being able to visually monitor cell growth across multiple days, and compare cell size and cell morphology between different cell lines.
Figure 1: A Schematic Diagram of the Three Different Methods Measuring Cell Proliferation. Cells were seeded at three different cell densities into multiple 96-well plates and incubated at 37 °C with 5% CO2 for up to 96 hr. Every 24 hr, cell proliferation was measured by either using a hemocytometer, a luminescence-based assay or cell imaging. Please click here to view a larger version of this figure.
Figure 2: Cell Proliferation Measurements after 96 hr. Cell counts were measured every 24 hr for 96 hr in MCF-7-LeGO and MCF-7-Δ40p53 cells seeded at three different cell densities. Cell counts were measured using a (a) hemocytometer in cells/ml, (b) using a luminescence-based assay in relative luminescent units (RLU), or (c) a continuous cell count using a cell imager in cells/image. Conditions for the cell imager are shown in Table 2. All experiments represent the mean of three independent experiments, ± the S.D. Please click here to view a larger version of this figure.
Figure 3: Comparison of Three Different Methods Measuring Cell Proliferation in Breast Cancer Cell Lines. A linear regression analysis was performed to compare the correlative relationships between the different methods examined for measuring cell proliferation in (a) MCF-7-LeGO cells and (b) MCF-7-Δ40p53 cells. A Pearson's correlation coefficient was calculated and the significance was determined (p <0.05) between the different methods measuring cell proliferation. All results represent the mean ± the S.D of three independent experiments. Please click here to view a larger version of this figure.
Figure 4: Images of GFP-positive MCF-7-LeGO and MCF-7-Δ40p53 Breast Cancer Cells. Representative images of cells from 5 x 103 seeded cells captured using a cell imager every 24 hr. The scale bar represents 1,000 µm. Parameters for cell imaging are summarized in Table 2. Please click here to view a larger version of this figure.
Cell density (cells/well) | |||
Cell type | 1.5 x 103 | 3.0 x 103 | 5 x 103 |
MCF-7-LeGO | 76 µl in 8.4 ml media | 152 µl in 8.3 ml media | 254 µl in 8.2 ml media |
MCF-7-Δ40p53 | 93 µl in 8.4 ml media | 185 µl in 8.3 ml media | 308 µl in 8.2 ml media |
Table 1: Cell Seeding Volumes for 96 well plates.
Read parameters | |
Plate type | 96 well |
Mode | Image |
Objective | 2.5X |
Color | GFP (469, 525), Bright Field |
Exposure | Auto |
Focus | Auto (Scan then auto focus) |
Table 2: Read Parameters for Cell Imaging Using the Cell Imager.
Imaging parameters | |
Threshold | 5000 |
Minimum object size (µm) | 30 |
Maximum object size (µm) | 300 |
Table 3: Imaging Parameters for Cell Counting Analysis.
MCF-7-LeGO | ||
R square | p-value | |
Hemocytometer vs. luminescence-based assay | 0.6301 | 0.0021 |
Cell imager vs. hemocytometer | 0.7524 | 0.0003 |
Luminescence-based assay vs. cell imager | 0.8899 | < 0.0001 |
MCF-7-Δ40p53 | ||
R square | p-value | |
Hemocytometer vs. luminescence-based assay | 0.8983 | < 0.0001 |
Cell imager vs. hemocytometer | 0.9303 | < 0.0001 |
Luminescence-based assay vs. cell imager | 0.9805 | < 0.0001 |
Table 4: Linear Regression Analysis of the Three Different Proliferation Methods Tested.
Method | Advantages | Disadvantages | Technical notes | Final output |
Hemocytometer | Low cost | High human error | Pipette multiple times to prepare single cell suspension | Cells/ml |
Requires minimal equipment | Requires single-cell suspension | Perform multiple counts to achieve accuracy | ||
Direct cell count | High number of cells required for accurate assessment of cell count | |||
Endpoint | ||||
Luminescence-based assay | Use with multiwell-plate formats | Expensive reagents | Protect from light | Relative Luminescent Units (RLU)/well |
Easy to perform | Requires luminescent plate reader | Include control wells to determine background luminescence | ||
Fast assay | Temperature-sensitive | |||
Provides cell viability information | Variable depending on metabolic activity of cells | |||
Indirect measurement | ||||
Endpoint | ||||
Cell imager | Continuous measurement | Expensive imager | Ensure cell imager is set to 37 °C | Cells/image |
Temperature control | Skill intensive | Avoid unnecessary shaking or disruption of cells | ||
Provides cellular information | Variable depending on confluence of cells | |||
Cost-effective (if you have the imager) | Relative count | |||
Direct measurement | ||||
Automated imaging of multiwell-plate format |
Table 5: Comparison of the Advantages and Disadvantages of the Different Cell Counting Methods.
In this protocol three different methods of measuring cell proliferation in cultured cells were examined. Each method was capable of reproducible and accurate measurements of cell proliferation over 96 hr, and the results were comparable between each of the methods tested (Figure 2 and 3). Both the luminescence-based assay and cell imaging method produced the most robust results, showing linear increases in cell proliferation after 96 hr (Figure 2b, c). Additionally, cell imaging over time depicted no significant difference in the growth rates between the transduced and non-transduced cell lines (Figure 4).
There are many advantages and disadvantages for each method examined in this protocol, see Table 5 for a summary. The conventional cell counting method using a hemocytometer is a low cost method that requires very little additional reagents or effort to prepare and run. Furthermore, this method quantitates an absolute cell count in cells/ml14. However, there are serious disadvantages, which include the time consuming nature of the cell counting, high error rates that results in large standard deviations between counts, and the fact that a high range of cell numbers are necessary for accurate cell counts. This can be seen in Figure 2a, where cell counting using the hemocytometer showed variable results at the low cell densities, and large standard deviations at the later time points. These disadvantages make this method useful for cell counting of small sample sizes, and inadequate for larger high throughput measurements where smaller plate sizes and seeding densities are required. These limitations could be alleviated if the cell density was increased, such that the minimum number of cells counted began at a threshold of greater than 100 cells. The more diluted the cell suspension, or lower the cell density, the greater chance of counting less than 100 cells and therefore increasing the variability between replicates15. However, this method is unsuitable for a 96 well plate, due to the low cell surface area, and hence, the insufficient number of cells that can be used in the analysis. This highlights the lack of high throughput capabilities of this method and a clear disadvantage for users who require this capability.
The luminescence-based assay determines cell viability by measuring the amount of ATP, which is a measure of the presence of metabolically active cells16. This assay is designed for high throughput screening of multiple samples in a 96 well plate format to determine cell proliferation. This simple method quantitates cell proliferation as a relative luminescence unit (RLU) using a plate reader, which is proportional to the ATP present in the metabolically active cells. However, the major disadvantage of this method is the cost of the reagent, and the dependence of the measurement on the metabolic activity of the cells. Various cell culture conditions, such as temperature or cell cycle time points, can readily affect the amount of ATP produced by the cells, which is directly proportional to the luminescent signal generated by the assay17. Therefore, it is important to empirically determine that the conditions of the experiment do not interfere with the metabolic activity of the cells and potentially impede the relative luminescent signal generated by the cells.
The third method examined for determining cell proliferation was a live cell imager and software to perform a relative cell count. The cell imager has high-throughput capabilities as it can be automated to capture multiple images for each well, in real-time along with temperature control, using the same cells over the duration of the assay. As shown in Figure 4, cell proliferation can be monitored in the same plate over a time course, which can provide additional information about the cell population along with cell counting analysis. This is very advantageous as it eliminates cross-plate variability and cell seeding error, which is commonplace in 96 well plate formats. The software accompanying the cell imager allows for the quantitation of cell counts using the parameters outlined in Table 2 and 3. It is therefore useful in a high-throughput setting and can easily be multiplexed to other assays to measure cellular functions such as apoptosis or cytotoxicity. A major disadvantage of the system is the software, which is limited in its ability to split touching objects. While it can perform this function in lower confluence cells, it is a major limitation of the assay and therefore requires cell-type specific optimization. Also, while individual experiments using an imager are very low cost on a plate-by-plate scale, this method would only be cost effective if the instrument is already set up in a laboratory. Therefore, this provides a novel method for measuring cell proliferation of cultured cells, and has the major advantage of requiring no additional reagents, and is capable of 96 well plate automated counting, making this method appropriate for high-throughput analysis. This is a significant improvement on the conventional hemocytometer cell counting method, and is a more cost effective option to the luminescence-based assay.
The critical step when using the cell imager is during the analysis of the captured images and subsequent cell count per image. These images can be processed using a number of cell imaging platforms. It is therefore paramount to determine the most appropriate software for the cell type under investigation. It would be useful to validate the cell counts from the images acquired by the cell imager using different imaging software. This protocol could be applied to any cultured cells, however the analysis parameters would have to be defined for each cell line. This can be time consuming at first, but once the parameters have been set, the method can be automated to image whole plates at a time.
It is important to note that these assays are in fact an indirect measure of proliferation, as they measure cell number (hemocytometer, cell imager), or metabolic activity (luminescence-based assay), over a period of time. These methods can be easily amended to measure other important factors that can influence proliferation. For example, the trypan blue exclusion method can easily be incorporated into either the hemocytometer or cell imager method to exclude dead cells and hence determine cell viability13. The luminescence-based assay can be multiplexed with a cytotoxicity assay to provide additional information about cell health, and the metabolic activity measured during this assay is also a measurement of cell viability12. Therefore, the flexibility of these assays to be multiplexed with other assays to provide additional cellular information is a strong advantage of each of these methods.
This protocol used the human breast cancer cell line MCF-7 which have been stably transduced with the LeGO-iG2-puro+ vector containing the GFP gene. This allows the use of the GFP optics filter to image the cells, without the need to add any dye agents into the culture medium. This method can easily be modified to image GFP-negative cells or even primary cell lines, either by imaging the cells using the bright field optics type, or by labelling the cells with a proliferation marker. The methods compared in this protocol are robust enough to be used in a wide variety of different cell lines, however the optimal protocol settings for each individual cell line should be determined first. Imaging of cells using the bright field channel represents the best option for primary cells or those that are slow growing, due to the long-term nature of this method. End-point assays, such as the luminescence-based assay, are not well-suited for the analysis of long-term cell growth.
This protocol has described three different methods for measuring cell proliferation in vitro. Each method can be routinely performed in the laboratory to measure cell growth, and most laboratories would possess the necessary skills to perform one of these methods. However, there are also a range of other assays available for measuring dividing cells. These can include methods that measure DNA synthesis, metabolic activity, associated antigens of proliferation or ATP concentration9. For example, a number of assays have been developed to measure the rate of DNA synthesis of cells by labelling cells with a radioactive substance such as 3H-thymidine. A drawback of this method is the obvious use of radioactive substances, and their disposal. Other methods which are routinely used for measuring cell proliferation include the use of BrdU labelling of newly formed DNA, which can be measured using flow cytometry18. Flow cytometry can be used to analyze both cell number as well as cell cycle information. This may be an advantageous outcome for particular experiments, however the disadvantages of this method include the additional time and steps, expensive reagents and increased user-trained skills in order to operate the flow cytometer and analyze the resulting data18. Therefore, there are many different assays available to scientists to evaluate the growth of cells, and the chosen method is largely dependent on the cell type used, the user and their skill level, and the type of data required at the endpoint of the experiment.
In conclusion, three different methods of measuring cell proliferation were compared using breast cancer cells. Each method has many advantages and disadvantages, and is therefore user-dependent and based on their requirements for each experiment, in terms of determining the most appropriate assay to perform cell counting.
The authors have nothing to disclose.
We would like to thank Dr Hamish Campbell and Prof Antony Braithwaite for their help in developing the transduced MCF-7-LeGO cell lines. We would like to acknowledge our funding support by the Bloomfield Group Foundation through the Hunter Medical Research Institute. B.C.M is supported by an APA scholarship through the University of Newcastle and the MM Sawyer Scholarship through the Hunter Medical Research Institute.
Dulbecco's Modified Eagle Medium, no phenol-red | ThermoFisher Scientific | 21063-045 | Supplemented with 10% FBS, 200mM L-glutamine, 2µg/ml insulin and 1µg/ml puromycin |
L-glutamine solution (100x) | ThermoFisher Scientific | 25030-081 | |
Insulin solution human | Sigma-Aldrich | I9278-5ML | |
Fetal bovine serum (FBS) | Bovogen Biologicals | SFBS-F-500ml | |
Puromycin dihydrochloride | Sigma-Aldrich | P9620-10ML | |
0.5% trypsin-EDTA solution (10x) | ThermoFisher Scientific | 15400-054 | Dilute to 2x in DPBS |
Dulbecco's Phosphate Buffered Saline (DPBS) (1x) | ThermoFisher Scientific | 30028-02 | |
Tissue culture flask, 75cm2 growth area | Greiner Bio-One | 658175 | |
Scepter 2.0 Cell Counter | Merck Millipore | Automated cell counter | |
96 well multiwell plate, flat bottom | Nunc | 167008 | |
Improved Neubauer Hemocytometer | BOECO Germany | BOE 01 | |
Olympus IX51 inverted microscope | Olympus | IX51 | |
CellTiter-Glo 2.0 Assay | Promega | G9242 | Luminescence-based assay |
Cytation 3 Cell Imaging Multi-Mode Reader | BioTek | Plate reader for luminescence, fluorescence and brightfield cell imaging | |
Gen5 Data Analysis Software | BioTek | GEN5 |