Name: comparison of three different methods for determining cell proliferation in breast cancer cell lines
Uploaded: 2016-09-03T13:00:00
Description: Watch this Scientific Journal Video about Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines at JoVE.com

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% CO<...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium, no phenol-red</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21063-045</td>
			<td style="width:597px;">Supplemented with 10% FBS, 200mM L-glutamine, 2&#181;g/ml insulin and 1&#181;g/ml puromycin</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">L-glutamine solution (100x)</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin solution human</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9278-5ML</td>
			<td style="width:597px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fetal bovine serum (FBS)</td>
			<td style="width:99px;">Bovogen Biologicals</td>
			<td style="width:119px;">SFBS-F-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620-10ML</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.5% trypsin-EDTA solution (10x)</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15400-054</td>
			<td>Dilute to 2x in DPBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (1x)</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">30028-02</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:216px;">Tissue culture flask, 75cm2 growth area</td>
			<td style="width:99px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">658175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scepter 2.0 Cell Counter</td>
			<td style="width:99px;">Merck Millipore</td>
			<td style="width:119px;"></td>
			<td>Automated cell counter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96 well multiwell plate, flat bottom</td>
			<td style="width:99px;">Nunc</td>
			<td align="right" style="width:119px;">167008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Improved Neubauer Hemocytometer</td>
			<td style="width:99px;">BOECO Germany</td>
			<td style="width:119px;">BOE 01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus IX51 inverted microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:119px;">IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CellTiter-Glo 2.0 Assay</td>
			<td style="width:99px;">Promega</td>
			<td style="width:119px;">G9242</td>
			<td>Luminescence-based assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytation 3 Cell Imaging Multi-Mode Reader</td>
			<td style="width:99px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>Plate reader for luminescence, fluorescence and brightfield cell imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gen5 Data Analysis Software</td>
			<td style="width:99px;">BioTek</td>
			<td style="width:119px;">GEN5</td>
			<td></td>
		</tr>
	</tbody>
</table>

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-&#916;40p53 transduced cells was compared to the non-transduced MCF-7-LeGO breast cancer cell line. The three methods that were compared&#160;&#8211; 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...

Watch this Scientific Journal Video about Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines at JoVE.com

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

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 ...

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