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.

増殖アッセイのために1.準備細胞注：各メソッドを分析するために同じ形式で同じ方法及び種子中の2つの細胞株を準備します。 <ol><li> 10％ウシ胎児血清（FBS）を補充したフェノールレッドフリーのダルベッコ改変イーグル培地（DMEM）を用いてT 75cm 2の組織培養フラスコ中で75％〜80％コンフルエンスに7個の細胞を、MCF-7、レゴとMCF-7Δ40p53成長、200...

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このプロトコルでは、培養細胞における細胞増殖を測定する3つの異なる方法を検討しました。それぞれの方法は、96時間にわたる細胞増殖の再現性と正確な測定が可能であった、その結果は（ 図2及び3）試験方法の各々の間で同等でした。発光ベースのアッセイ及び細胞イメージング法の両方が、96時間後の細胞増殖の線形増加を示し、最も堅牢な結果が得られた（ 図<stron...

腫瘍抑制遺伝子は、p53は、細胞周期停止、アポトーシス及び老化1を含む細胞プロセスの多数の重要な調節因子です。これは、ゲノムの安定性を維持する責任があり、したがって、細胞死と細胞増殖のバランスを維持するために重要です。 p53の突然変異は、癌において一般的であり、制御されていない癌細胞の増殖を2につながるp53の不活性化の主な原因です。興味深いことに、p53の中の変異は、唯一の他のメカニズムがp53機能の喪失の原因であることを示唆し、乳癌3の約25％を占めています。最近発見されたp53のアイソフォームは、ヒト癌の多数において過剰発現されることが示されており、p53機能4,5を調節することができます。我々は、p53のアイソフォーム、Δ40p53は、乳癌において最も高度に発現されるアイソフォームであることが以前に示されており、正常な隣接TISSと比較した場合に有意に、乳癌細胞において上方制御されますUE 6。その後、我々は、安定レゴ-IG2-puroを+ベクターを用いΔ40p53（GFP +）の7を過剰発現するMCF-7ヒト乳癌細胞株を形質導入しました。これらの細胞は高いΔ40p53発現が乳癌細胞の細胞増殖率を増加させるかどうかを調べるために使用しました。 インビトロ 8,9 で培養した細胞の細胞増殖を測定する多くの直接的及び間接的な方法があります。これらは、時間の経過とともに連続測定として、またはエンドポイントアッセイ10のいずれかに行うことができます。従来の方法は、血球計数器を用いて細胞計数として、依然として有用です。このアッセイは、低コストと細胞数の直接の尺度であるが、それは数からの誤差と大きな標準偏差を最小にするために大規模な細胞数と高度に熟練した訓練に依存しません。ハイスループットフォーマットと互換性のある測定を行う必要がマルチウェルプレートアッセイの開発につながっています。これらの発光ベースアッセイmeasuセル11,12の代謝活性に比例する発光シグナルに基づいて、細胞数の再。より最近では、高含量のイメージングプラットフォームの導入は、定量的及び定性的な表現型データ収集を提供しつつ、細胞増殖をモニターする新しいツールを許可し、システム13の様々な含まれています。これらの方法の全ては、連続測定またはエンドポイントアッセイのいずれかによって、細胞増殖を測定するための手段を提供し、それぞれの感度、サンプル番号のスループット、およびセル情報に関して利点と欠点の範囲を有し、によって適宜計量することができるすべてが研究の質問に。 このプロトコルは、それぞれの方法は、感度、再現性、およびマルチウェルプレートフォーマットの異なる範囲を用いて、in vitroで細胞増殖を測定するための3つの異なる方法を記載しています。このプロトコルは、茶をカウント血球計数器の使用を比較することを目的としましたmber、96時間の時間経過にわたって細胞増殖の測定における発光に基づく細胞生存率アッセイ、および細胞イメージャ。これを行うには、ベクター形質導入細胞（MCF-7-LEGO）の成長は、3つの異なる細胞密度を使用して、Δ40p53（MCF-7-Δ40p53）を過剰発現するように形質導入​​された細胞と比較しました。細胞増殖は、最大96時間ごとに24時間測定しました。それぞれの方法は、それ自身の利点と欠点を有することが見出され、実験の目的に応じて、各々が依然として増殖の速度についての情報を提供するための有益な方法で行いました。

<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.

培養された細胞の増殖を測定する別の方法を研究するために、MCF-7Δ40p53形質導入細胞の細胞増殖は、非形質MCF-7レゴ乳癌細胞株と比較しました。比較された三つの方法-従来の血球計数法、細胞生存率発光アッセイ、及び概略図（ 図1）に概説されている解析-細胞イメージング。それぞれの方法にも長所と短所が正確に時間をかけて細胞計数を測定するこ?...

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.

乳癌細胞株における細胞増殖を決定するための3つの異なる方法の比較

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, carefully aspirate and dispose of the supernatant. Afterward, re-suspend the cell pellet in five milliliters of fresh supplemented DMEM.To perform a cell count, dilute 100 microliters of the cell suspension in 900 microliters of one times DPBS. Then, place a new 60-micrometer 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. Then, remove the sensor from the cell counter when completed. Seed the cells in a final volume of 100 microliters in a 96-well plate at about 20%confluency to allow room for cell growth and measurement of proliferation over three to five days.To determine the cell count using a hemocytometer, pre-warm both the medium and trypsin to 37 degrees Celsius in a cell culture incubator, oven, or water bath. Next, aspirate the medium from the cells into a waste container. Then, wash them once with 30 microliters of two times trypsin and aspirate the medium into the waste container again.Next, wash the cells again with 30 microliters of two times trypsin, and incubate them for five minutes at 37 degrees Celsius. Gently tap the plate edge to dislodge the cells. Subsequently, add 50 microliters of supplemented DMEM and mix the cells by pipetting until a single cell suspension is formed.Place the glass cover slip over the counting chambers and affix it until Newton's refraction rings are seen between the cover slip edges and the helocytometer. Then, gently pipette 20 microliters of the cell supension under the cover slip and allow the counting chamber to be filled by capillary motion. Subsequently, visualize the counting chambers in the grid layout under 10x magnification.In this procedure, thaw the luminescence reagent in a 22 degrees Celsius water bath for 30 minutes. Gently invert the bottle to obtain a homogeneous mix. Then, equilibrate one plate of seeded cells at room temperature for 30 minutes.Afterward, add 100 microliters of luminescence reagent to each well. Mix the contents on an orbital shaker for two minutes. Then, allow the plate to incubate for 10 minutes at room temperature.To set up a luminescence experiment using the multi-mode plate reader software, turn on the multi-mode plate reader and open the software. In the Task Manager, select Experiments and Create New. Then, select File from the side toolbar, and under the Protocol tab, select Procedure.Next, select Greiner 96 flat bottom as the plate type, and check the Use Lid box. Select the Read action under the Read method box. Then, select Luminescence as the detection method and click OK.In the Read Step box, select the wells to be scanned under the Full Plate tab, and select wells to act as blank wells.Set the Filter Set to Empty Filter. Set the Gain to 135, with an Integration Time of 5 seconds per well and a Read Height of 6.5 millimeters. Click OK to save the settings for the luminescence read.To perform a luminescence read, eject the plate holder by selecting the Instrument Control tab and click Plate Out. Place the 96-well plate onto the plate holder, ensuring the lid is on. Close the plate holder by clicking on Plate In under the Instrument Control tab.Then, click Read Now in the toolbar to perform a luminescent read. After that, export the RLU measurements for each well to a spreadsheet for further analysis. To set up a cell imaging experiment, turn on the multi-mode cell imager and open the software.In the Task Manager, select the Experiments tab and Create New. On the File toolbar, click on the Protocol tab and then the Procedure tab. Select the Set Temperature action.Set the Incubator to On, and set the Temperature to 37 degrees Celsius. Check Preheat, then click OK to save the settings. Next, select the Read action.Click on Image as the detection method. Then, select the Endpoint/Kinetic read type and the Filters optics type, and click OK.After that, click the Full Plate tab, and select the wells on the plate to be imaged. Click OK to save the settings.Now, select the 2.5 times objective from the drop-down Objectives option. Under the Channels tab, select GFP 469.525 and Bright Field. Check Auto Exposure for both channels, and select the auto exposure well.To determine auto focus settings, click Options. For the auto focus options, select the method Scan and then auto focus. Click OK to save the settings.After that, set the horizontal and vertical offset from the center of the well to zero. Select to scan multiple images per well in a three by two montage. Then, click OK to save the procedure settings.To read the experiment on the selected plate, click on the Instrument Control tab and select the Plate Out function. Place a 96-well plate on the plate holder, ensuring the lid is on. Under the Instrument Control tab, select the Plate In function.Next, select Read Now from the plate tab, and repeat the read every 24 hours, up to 96 hours post-seeding. To analyze an image, click on the Data tab on the imaged plate and select Picture GFP 469, 525 plus Bright Field. Then, double-click on the imaged well.Now, click on a loaded image. Select Analyze, and then select the single image of the montage to be analyzed. Next, click OK.Afterward, check the GFP channel only, and set the parameters as outlined in Table 3.After that, click Start to the apply the parameters to the imaged cells. 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.In this figure, a linear regression analysis was performed to compare the correlative relationships between the different methods examined for measuring the cell proliferation in MCF-7-LeGO cells and MCF-7-delta 40p53 cells. A Pearson's correlation coefficient was calculated, and the significance was determined between the different methods measuring cell proliferation. The strongest correlation was observed between the comparison of the luminescence-based assay and the cell imager.Shown here are the representative images of GFP positive MCF-7-LeGO and MCF-7-delta 40p53 breast cancer cells captured using the cell imager every 24 hours until the cells reach close to 100%confluence. 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. A summary of the advantages and disadvantages of each method tested is shown here, which demonstrates the versatility of each of the methods and will assist readers in choosing the most appropriate method.After watching this video, you should have a good understanding of three different cell proliferation assays, and should be able to determine which one best suits your own experimental design.

Research

JoVE Journal

Biology

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

トリプシンで継代色合いヒト胚性幹細胞、線

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

三次元細胞培養用チップサイズの足場の微細加工

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

セプターのセルカウンターを用いて細胞培養中の細胞数の決定

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

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

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

テトラサイクリン誘導性（テトオン）shRNAまたはcDNA発現で安定ヒト細胞株の世代

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

創傷応答のためのスキン傷つけ処理のための方法およびアッセイ<em&gt; C。エレガンス</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

CRISPR / Cas9経由哺乳動物細胞株におけるゲノム欠失の生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

細胞株および組織試料中の脱ユビキチン化酵素の活性を測定する方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ホルマリン固定パラフィン包埋参照標準細胞株にBRAF V600E変異を検出するデジタルドロップレットPCRを採用

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

無傷の植物をブランチングすることによって、または抽出物の熱処理によりタバコ宿主細胞タンパク質の除去方法の比較

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...