Name: Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines (Video) | JoVE Journal
Uploaded: 2016-09-03T13:00:00.000+00:00
Duration: 12 min 35 s
Description: Learn to compare three cell proliferation assays for breast cancer research using MCF-7 cell lines. This protocol covers conventional counting, luminescence-based viability, and cell imager methods with detailed setup steps.

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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.

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.
<ol>
	<li>Grow MCF-7-LeGO and MCF-7-&#916;40p53 7 cells to 75-80% confluence in T 75 cm2 tissue culture flasks using phenol-red free Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 2 &#181;g/ml insulin and 1 &#181;g/ml Puromycin at 37 &#176;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.</li>
	<li>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 &#176;C with 5% CO2.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Dilute 100 &#181;l of the cell suspension in 900 &#181;l 1x Dulbecco&#39;s Phosphate Buffered Saline (DPBS). Place a new 60 &#181;m 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.</li>
	</ol>
	</li>
	<li>Seed cells at a final volume of 100 &#181;l (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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Maintain cells at 37 &#176;C with 5% CO2 for 24 hr.</li>
</ol>
2. Determining Cell Count Using a Hemocytometer
<ol>
	<li>Pre-warm both medium and trypsin to 37 &#176;C in a cell culture incubator, oven or water bath. Aspirate media from cells into a waste container, wash once with 30 &#181;l of 2x trypsin, and aspirate the trypsin.</li>
	<li>Wash cells again with 30 &#181;l of 2x trypsin, and incubate for 5 minutes at 37 &#176;C. Gently tap edge of plate to dislodge cells from the plate. Add 50 &#181;l of supplemented DMEM and mix cells by pipetting until the cells form a single cell suspension.</li>
	<li>Prepare hemocytometer by cleaning surface and glass cover with 70% ethanol.</li>
	<li>Place the glass cover-slip over counting chambers and affix until &#39;Newton&#39;s refraction rings&#39; can be seen between the cover-slip edges and the hemocytometer. 
	Note: These indicate that the cover-slip has correctly adhered to the hemocytometer.</li>
	<li>Gently pipette 20 &#181;l 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.</li>
	<li>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 cells14,15.
	<ol>
		<li>Calculate cell concentration per ml as follows:Average cell count per square x dilution factor (if used) x 104</li>
	</ol>
	</li>
	<li>Repeat steps 2.1-2.8 every 24 hr for 96 hr post-seeding.</li>
</ol>
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.
<ol>
	<li>Thaw luminescence reagent in a 22 &#176;C water bath for 30 min.</li>
	<li>Gently mix reagent by inverting the bottle to obtain a homogenous mix.</li>
	<li>Equilibrate one plate of seeded cells (from step 1.5) at RT for 30 min.</li>
	<li>Add 100 &#181;l of luminescence reagent to each well. Mix contents on an orbital shaker for 2 min. Allow plate to incubate for 10 min at RT.</li>
	<li>Setting up a luminescence experiment using the multi-mode plate reader software.
	<ol>
		<li>Turn on multi-mode plate reader and open the software. In the &#39;task manager&#39;, select &#39;experiments&#39; and &#39;create new&#39;.Select &#39;file&#39; from side toolbar, and under &#39;protocol&#39; tab, select &#39;procedure&#39;.</li>
		<li>Select &#39;Grenier 96 flat bottom&#39; plate type, and check the &#39;use lid&#39; box. Select &#39;read&#39; action under &#39;read method&#39; box. Select &#39;luminescence&#39; detection method. Click &#39;OK&#39;.</li>
		<li>In the read step box, select the wells to be scanned under the &#39;full plate&#39; tab, and select wells to act as blank wells.</li>
		<li>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 &#39;OK&#39; to save settings for the luminescence read.</li>
	</ol>
	</li>
	<li>Performing a Luminescent Read
	<ol>
		<li>Eject plate holder by selecting &#39;instrument control&#39; tab and click on &#39;plate out&#39;.Place 96-well plate onto the plate holder, ensuring the lid is on. Close the plate holder by clicking on &#39;plate in&#39; under &#39;instrument control&#39; tab. Click on &#39;read now&#39; from toolbar to perform luminescent read.</li>
		<li>Export the relative luminescent unit (RLU) measurements for each well to a spreadsheet for further analysis.</li>
	</ol>
	</li>
	<li>Repeat steps 3.1-3.6 to record proliferation every 24 hr up to 96 hr after seeding cells.</li>
</ol>
4. Determining Cell Count Using a Cell Imager
<ol>
	<li>Setting up a cell imaging experiment using the multi-mode cell imager software
	<ol>
		<li>Turn on multi-mode cell imager and open the software.</li>
		<li>In the &#39;task manager&#39;, select &#39;experiments&#39; tab and &#39;create new&#39;.On the &#39;file&#39; toolbar, click on &#39;protocol&#39; tab and open &#39;procedure&#39; tab.Select &#39;set temperature&#39; action. Set incubator to &#39;on&#39; and set temperature to 37 &#176;C and check &#39;preheat&#39; before continuing with next step&#39; box. Click &#39;OK&#39; to save settings.</li>
		<li>Select &#39;read&#39; action. Click on &#39;image&#39; detection method, select &#39;endpoint/kinetic&#39; read type and &#39;filters&#39; optics type. Click &#39;OK&#39;.Click on &#39;full plate&#39; tab and select wells on the plate to be imaged. Click &#39;OK&#39; to save the settings.</li>
		<li>Select the 2.5X objective from the drop down &#39;objectives&#39; options. Under &#39;channels&#39; tab, select two channels: GFP 469, 525 and Bright Field. Check &#39;auto&#39; exposure for both channels and select the auto-exposure well.</li>
		<li>To determine auto focus settings, select &#39;auto-focus&#39; and click on &#39;options&#39;. For the auto-focus options, select the &#39;scan and then auto focus&#39; method. Click &#39;OK&#39; to save settings.</li>
		<li>Set the horizontal and vertical offset from center of well (&#181;m) to 0.Select to scan multiple images per well in a 3 x 2 montage, with horizontal spacing (&#181;m) = 2,881 and vertical spacing (&#181;m) = 2,127. Click &#39;OK&#39; to save procedure settings. 
		Note: See Table 2 for read parameters.</li>
	</ol>
	</li>
	<li>Performing Read Experiment on Selected Plate
	<ol>
		<li>Click on the &#39;instrument control&#39; tab and select the &#39;plate out&#39; function.Place 96-well plate in plate holder, ensuring the lid is on. Under the &#39;instrument control&#39; tab, select the &#39;plate in&#39; function. Select &#39;read now&#39; from plate tab. Repeat read every 24 hr up to 96 hr post-seeding.</li>
	</ol>
	</li>
	<li>Analysis of Cell Count Using the Cell Imager Software.
	<ol>
		<li>To analyze an image, click on the &#39;data&#39; tab on the imaged plate, select &#39;picture [GFP 469, 525 + Bright field]&#39;. Double click on an imaged well.</li>
		<li>Click on a loaded image. Select &#39;analyze&#39; and select a single image of the montage to be analyzed. Click &#39;OK&#39;.Check the &#39;GFP&#39; channel only, and set parameters as outlined in Table 3. Click &#39;START&#39; to apply parameters to the imaged cells.</li>
		<li>Observe the cell counting mask placed over the imaged cells, which is used to determine the cell count per image. Click &#39;apply changes&#39; and maintain these settings for each plate imaged throughout the experiment.</li>
		<li>Once back at the imaged plate, click on the &#39;data&#39; drop down menu and select &#39;cell count&#39;. This will generate the total cell count per well.</li>
		<li>Export all data to a spreadsheet by clicking on the &#39;export&#39; tab for further analysis of the cell counts per well.</li>
	</ol>
	</li>
</ol>

<ol><li>Lane, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer.+p53%2C+guardian+of+the+genome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cancer. p53, guardian of the genome.</a> Nature. 358 (6381), 15-16 (1992).</li>
<li>Olivier, M., Hollstein, M., Hainaut, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TP53+mutations+in+human+cancers%3A+origins%2C+consequences%2C+and+clinical+use%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">TP53 mutations in human cancers: origins, consequences, and clinical use.</a> Cold Spring Harb Perspect Biol. 2 (1), a001008(2010).</li>
<li>Olivier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+clinical+value+of+somatic+TP53+gene+mutations+in+1%2C794+patients+with+breast+cancer%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer.</a> Clin Cancer Res. 12 (4), 1157-1167 (2006).</li>
<li>Bourdon, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((p53+isoforms+can+regulate+p53+transcriptional+activity%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">p53 isoforms can regulate p53 transcriptional activity.</a> Genes Dev. 19 (18), 2122-2137 (2005).</li>
<li>Avery-Kiejda, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecular+weight+variants+of+p53+are+expressed+in+human+melanoma+cells+and+are+induced+by+the+DNA-damaging+agent+cisplatin%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin.</a> Clin Cancer Res. 14 (6), 1659-1668 (2008).</li>
<li>Avery-Kiejda, K. A., Morten, B., Wong-Brown, M. W., Mathe, A., Scott, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relative+mRNA+expression+of+p53+isoforms+in+breast+cancer+is+associated+with+clinical+features+and+outcome%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome.</a> Carcinogenesis. 35 (3), 586-596 (2014).</li>
<li>Weber, K., Bartsch, U., Stocking, C., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multicolor+panel+of+novel+lentiviral+%26%2334%3Bgene+ontology%26%2334%3B+%28LeGO%29+vectors+for+functional+gene+analysis%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">A multicolor panel of novel lentiviral &#34;gene ontology&#34; (LeGO) vectors for functional gene analysis.</a> Mol Ther. 16 (4), 698-706 (2008).</li>
<li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRiss%2C+TL+%22Cell+Biology%22">Cell Biology</a>. Celis, J. E. , Third Edition, Academic Press. 25-31 (2006).</li>
<li>Ng, K. W., Leong, D. T., Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+challenge+to+measure+cell+proliferation+in+two+and+three+dimensions%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">The challenge to measure cell proliferation in two and three dimensions.</a> Tissue Eng. 11 (1-2), 182-191 (2005).</li>
<li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time%2C+dose+of+toxin%2C+and+plating+density+in+cell-based+cytotoxicity+assays%5BTitle%5D)+AND+%22Assay+Drug+Dev+Technol%22%5BJournal%5D)">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays.</a> Assay Drug Dev Technol. 2 (1), 51-62 (2004).</li>
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<li>Bastidas, O. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBastidas%2C+O+%22Cell+Counting+with+Neubauer+Chamber%3A+Basic+Hemocytometer+Usage%22">Cell Counting with Neubauer Chamber: Basic Hemocytometer Usage</a>. , 
 
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<li>Bastidas, O. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBastidas%2C+O+%22Technical+Note%3A+Cell+Count+for+Low+Concentration+Samples%0A++%22">Technical Note: Cell Count for Low Concentration Samples
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<li>Riss, T. L., Moravec, R., Niles, A. L., et al. Cell Viability Assays. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRiss%2C+TL+%22Assay+Guidance+Manual%5BInternet%5D%22">Assay Guidance Manual[Internet]</a>. Sittampalam, G. S., Coussens, N. P., Nelson, H., et al. , (2013 May 1 [Updated 2015 Jun 29]) 
 
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<li>Quent, V. M., Loessner, D., Friis, T., Reichert, J. C., Hutmacher, D. W. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aQuent%2C+VM+%22J+Cell+Mol+Med%22">J Cell Mol Med</a>. 14 (4), 1003-1013 (2010).</li>
<li>Crane, J., Mittar, D., Soni, D., McIntyre, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCrane%2C+J+%22Cell+Cycle+Analysis+Using+the+BD+BrdU+FITC+Assay+on+the+BD+FACSVerse+System%22">Cell Cycle Analysis Using the BD BrdU FITC Assay on the BD FACSVerse System</a>. , (2013 May 1 [Updated 2015 Jun 29]) 
 
 <a target="_blank" href="https://www.bdbiosciences.com/documents/BD_FACSVerse_CellCycleAnalysis_AppNote.pdf">https://www.bdbiosciences.com/documents/BD_FACSVerse_CellCycleAnalysis_AppNote.pdf</a> 1-12 (2011).</li>
</ol>

The authors declare that they have no competing financial interests.

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...

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, &#916;40p53, 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 &#916;40p53 using the LeGO-iG2-puro+ vector (GFP+)7. These cells were used to investigate if high &#916;40p53 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 &#916;40p53 (MCF-7-&#916;40p53), 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.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium, no phenol-red</td><td>ThermoFisher Scientific</td><td>21063-045</td><td>Supplemented with 10% FBS, 200 mM L-glutamine, 2 &amp;micro;g/ml insulin and 1 &amp;micro;g/ml puromycin</td></tr><tr><td>L-glutamine solution (100x)</td><td>ThermoFisher Scientific</td><td>25030-081</td><td></td></tr><tr><td>Insulin solution human</td><td>Sigma-Aldrich</td><td>I9278-5ML</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Bovogen Biologicals</td><td>SFBS-F-500ml</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma-Aldrich</td><td>P9620-10ML</td><td></td></tr><tr><td>0.5% trypsin-EDTA solution (10x)</td><td>ThermoFisher Scientific</td><td>15400-054</td><td>Dilute to 2x in DPBS</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS) (1x)</td><td>ThermoFisher Scientific</td><td>30028-02</td><td></td></tr><tr><td>Tissue culture flask, 75 cm&lt;sup&gt;2 &lt;/sup&gt;growth area</td><td>Greiner Bio-One</td><td>658175</td><td></td></tr><tr><td>Scepter 2.0 Cell Counter</td><td>Merck Millipore</td><td></td><td>Automated cell counter</td></tr><tr><td>96 well multiwell plate, flat bottom</td><td>Nunc</td><td>167008</td><td></td></tr><tr><td>Improved Neubauer Hemocytometer</td><td>BOECO Germany</td><td>BOE 01</td><td></td></tr><tr><td>Olympus IX51 inverted microscope</td><td>Olympus</td><td>IX51</td><td></td></tr><tr><td>CellTiter-Glo 2.0 Assay</td><td>Promega</td><td>G9242</td><td>Luminescence-based assay</td></tr><tr><td>Cytation 3 Cell Imaging Multi-Mode Reader</td><td>BioTek</td><td></td><td>Plate reader for luminescence, fluorescence and brightfield cell imaging</td></tr><tr><td>Gen5 Data Analysis Software</td><td>BioTek</td><td>GEN5</td><td></td></tr></tbody></table>

comparison of three different methods for determining cell proliferation in breast cancer cell lines

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

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.

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 o...

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 ...

The overall goal of this demonstration is to compare three different cell proliferation assays and present the advantages and disadvantages of each method. This method can help answer key questions in the cancer research field, such as the use of the most appropriate cell proliferation method. Preparation of this method is critical, as the cell imager steps are difficult to learn.They require you to focus on the cells before applying the appropriate cell counting mask. To begin this procedure, grow two MCF-7 cell lines for three days to 75 to 80%confluence in the T75 square centimeter tissue culture flasks using phenol red-free DMEM. After three days, remove the cells from the flasks by pouring off the medium into a waste container.Then, immediately wash the cell layer with two milliliters of pre-warmed two times trypsin. Aspirate the trypsin, and add another two milliliters of pre-warmed trypsin to the cell layer before placing it in an incubator. Once the cells have detached, wash them with 10 milliliters of warmed fresh supplemented DMEM.Then, transfer the cell suspension to a sterile 15 milliliter tube and centrifuge it for five minutes at room temperature. After five minutes,

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Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

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Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

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Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

This study evaluates the proliferative effects of senescence-associated secretory phenotype (SASP) factors from senescent HeLa cells on non-senescent HeLa cells using three in vitro models with real-time monitoring. The advantages and limitations of each method were systematically compared to better understand cell-cell interactions in cancer.

Evaluating the Effect of SASP Factors on the Proliferation of Cancer Cells Using a Comparative Analysis of Three Distinct Methodologies

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

Learn to compare three cell proliferation assays for breast cancer research using MCF-7 cell lines. This protocol covers conventional counting, luminescence-based viability, and cell imager methods with detailed setup steps.

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

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