Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 function^4,5. 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 vitro^8,9. 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 cell^11,12. 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.

Protokół

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.

1. Grow MCF-7-LeGO and MCF-7-Δ40p53 ⁷ cells to 75-80% confluence in T 75 cm² tissue culture flasks using phenol-red free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 2 µg/ml insulin and 1 µg/ml Puromycin at 37 °C with 5% CO₂. Handle cells in a sterile biosafety cabinet class II.
   Note: The amount of time it takes to grow the cells to confluence varies depending on the seeding dilution. In preparation for plating the cells for the proliferation assays, seed the cells at a 1:3 dilution in supplemented DMEM and maintain in normal growth conditions for 3 days until 75-80% confluency.
2. To remove the cells from the flask, pour off the media into a waste container. Immediately wash the cell layer with 2 ml of pre-warmed 2x trypsin. Aspirate trypsin. Add a further 2 ml of pre-warmed trypsin to the cell layer and place in an incubator for 5 min at 37 °C with 5% CO₂.
   1. Once the cells detach, wash the cells with 10 ml warmed fresh supplemented DMEM. Transfer the cell suspension to a sterile 15 ml tube and centrifuge at 491 x g for 5 min at RT. Carefully aspirate and dispose of the supernatant.
3. Resuspend the cell pellet in 5 ml of fresh supplemented DMEM. Perform a cell count to determine the correct density to seed the cells in 96-well plates.
   1. Dilute 100 µl of the cell suspension in 900 µl 1x Dulbecco's Phosphate Buffered Saline (DPBS). Place a new 60 µm sensor onto the automated cell counter. Hold down the plunger and submerge the sensor in the diluted cell suspension. Slowly release the plunger on the cell counter. Remove the sensor from the cell counter when completed.
      Note: The cell count will be displayed on the cell counter in cells/ml.
4. Seed cells at a final volume of 100 µl (in DMEM) in a 96-well plate at ~20% confluency to allow room for cell growth and measurement of proliferation over 3-5 days (see Table 1). Seed all cell lines in triplicate.
   Note: For this experiment, three different cell densities were assessed to determine the optimal cell density. The required cell numbers are summarized in Table 1. Seed cells at a lower density if measurement of more long-term growth is required.
   1. For the hemocytometer and luminescence-based assays, prepare one plate per time point (i.e. 4 plates per assay). For the cell imager assay, prepare only one plate for the duration of the experiment.
5. Maintain cells at 37 °C with 5% CO₂ for 24 hr.

2. Determining Cell Count Using a Hemocytometer

1. Pre-warm both medium and trypsin to 37 °C in a cell culture incubator, oven or water bath. Aspirate media from cells into a waste container, wash once with 30 µl of 2x trypsin, and aspirate the trypsin.
2. Wash cells again with 30 µl of 2x trypsin, and incubate for 5 minutes at 37 °C. Gently tap edge of plate to dislodge cells from the plate. Add 50 µl of supplemented DMEM and mix cells by pipetting until the cells form a single cell suspension.
3. Prepare hemocytometer by cleaning surface and glass cover with 70% ethanol.
4. Place the glass cover-slip over counting chambers and affix until 'Newton's refraction rings' can be seen between the cover-slip edges and the hemocytometer.
   Note: These indicate that the cover-slip has correctly adhered to the hemocytometer.
5. Gently pipette 20 µl of cell suspension under the cover-slip, filling the counting chamber by capillary motion. Place hemocytometer under 10x magnification of a microscope and visualize the counting chambers in the grid layout.
6. Count cells in the 4 outer squares of the grid layout. To improve accuracy, repeat counts of additional outer squares if required, or until the count has reached 70 - 100 cells^14,15.
   1. Calculate cell concentration per ml as follows:Average cell count per square x dilution factor (if used) x 10⁴
7. Repeat steps 2.1-2.8 every 24 hr for 96 hr post-seeding.

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.

1. Thaw luminescence reagent in a 22 °C water bath for 30 min.
2. Gently mix reagent by inverting the bottle to obtain a homogenous mix.
3. Equilibrate one plate of seeded cells (from step 1.5) at RT for 30 min.
4. Add 100 µl of luminescence reagent to each well. Mix contents on an orbital shaker for 2 min. Allow plate to incubate for 10 min at RT.
5. Setting up a luminescence experiment using the multi-mode plate reader software.
   1. Turn on multi-mode plate reader and open the software. In the 'task manager', select 'experiments' and 'create new'.Select 'file' from side toolbar, and under 'protocol' tab, select 'procedure'.
   2. Select 'Grenier 96 flat bottom' plate type, and check the 'use lid' box. Select 'read' action under 'read method' box. Select 'luminescence' detection method. Click 'OK'.
   3. In the read step box, select the wells to be scanned under the 'full plate' tab, and select wells to act as blank wells.
   4. Set the filter set to empty filter (e.g., plug, Em: hole, mirror: none). Set the gain to 135, with an integration time of 0.5 sec per well and a read height of 6.5 mm. Click 'OK' to save settings for the luminescence read.
6. Performing a Luminescent Read
   1. Eject plate holder by selecting 'instrument control' tab and click on 'plate out'.Place 96-well plate onto the plate holder, ensuring the lid is on. Close the plate holder by clicking on 'plate in' under 'instrument control' tab. Click on 'read now' from toolbar to perform luminescent read.
   2. Export the relative luminescent unit (RLU) measurements for each well to a spreadsheet for further analysis.
7. Repeat steps 3.1-3.6 to record proliferation every 24 hr up to 96 hr after seeding cells.

4. Determining Cell Count Using a Cell Imager

1. Setting up a cell imaging experiment using the multi-mode cell imager software
   1. Turn on multi-mode cell imager and open the software.
   2. In the 'task manager', select 'experiments' tab and 'create new'.On the 'file' toolbar, click on 'protocol' tab and open 'procedure' tab.Select 'set temperature' action. Set incubator to 'on' and set temperature to 37 °C and check 'preheat' before continuing with next step' box. Click 'OK' to save settings.
   3. Select 'read' action. Click on 'image' detection method, select 'endpoint/kinetic' read type and 'filters' optics type. Click 'OK'.Click on 'full plate' tab and select wells on the plate to be imaged. Click 'OK' to save the settings.
   4. Select the 2.5X objective from the drop down 'objectives' options. Under 'channels' tab, select two channels: GFP 469, 525 and Bright Field. Check 'auto' exposure for both channels and select the auto-exposure well.
   5. To determine auto focus settings, select 'auto-focus' and click on 'options'. For the auto-focus options, select the 'scan and then auto focus' method. Click 'OK' to save settings.
   6. Set the horizontal and vertical offset from center of well (µm) to 0.Select to scan multiple images per well in a 3 x 2 montage, with horizontal spacing (µm) = 2,881 and vertical spacing (µm) = 2,127. Click 'OK' to save procedure settings.
      Note: See Table 2 for read parameters.
2. Performing Read Experiment on Selected Plate
   1. Click on the 'instrument control' tab and select the 'plate out' function.Place 96-well plate in plate holder, ensuring the lid is on. Under the 'instrument control' tab, select the 'plate in' function. Select 'read now' from plate tab. Repeat read every 24 hr up to 96 hr post-seeding.
3. Analysis of Cell Count Using the Cell Imager Software.
   1. To analyze an image, click on the 'data' tab on the imaged plate, select 'picture [GFP 469, 525 + Bright field]'. Double click on an imaged well.
   2. Click on a loaded image. Select 'analyze' and select a single image of the montage to be analyzed. Click 'OK'.Check the 'GFP' channel only, and set parameters as outlined in Table 3. Click 'START' to apply parameters to the imaged cells.
   3. Observe the cell counting mask placed over the imaged cells, which is used to determine the cell count per image. Click 'apply changes' and maintain these settings for each plate imaged throughout the experiment.
   4. Once back at the imaged plate, click on the 'data' drop down menu and select 'cell count'. This will generate the total cell count per well.
   5. Export all data to a spreadsheet by clicking on the 'export' tab for further analysis of the cell counts per well.

Wyniki

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...

Dyskusje

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, cel...

Materiały

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium, no phenol-red	ThermoFisher Scientific	21063-045	Supplemented with 10% FBS, 200 mM L-glutamine, 2 µg/ml insulin and 1 µg/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, 75 cm2 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