Name: a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells
Uploaded: 2018-02-16T13:00:00.000+00:00
Duration: 12 min 48 s
Description: Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

作者は感謝して乳房がん研究振興財団と高度な医療研究財団のサポートを認めます。

ここで説明したすべてのメソッドは、ボストン子供病院制度バイオ セーフティ委員会によって承認されています。
1. セル準備
<ol><li>
KHOS A を融解し、-n 個のセル
	<ol>
<li>ウォーム アップ培地、すなわち、ダルベッコ修飾イーグル培 10% (巻/巻) 牛胎児血清 (FBS) と 1% (巻/巻) ペニシリン/ストレプトマイシン。</li>
		<li>液体窒素タンクからセルのセラム チューブを取る、暖かい水 37 ° C の水浴中にバイアルの底を浸し、解凍処理を加速するセラム チューブを軽く振る。汚染を避けるために水へのお問い合わせからセラム チューブのふたを保ちます。細胞を解凍すると、すぐに、70% エタノール溶液を噴霧し、バイアルを拭く、セラムを滅菌します。</li>
		<li>層流フードの極低温バイアルから細胞懸濁液をすぐに吸引し、優しく温めて 10 mL 培養液 (1.1.1 で説明) で中断します。5-7 分の約 200 x g で細胞を懸濁液を遠心します。</li>
		<li>培地 10 mL を温めて、細胞ペレットを再懸濁します、T75 フラスコに移します。軽く均等に 10 mL のピペットを使用してセルを中断する数回をピペットします。</li>
		<li>5% (巻/巻) CO2と加湿雰囲気 37 ° C の定温器にフラスコを配置します。細胞の少なくとも 12 時間の接続を許可します。</li>
		<li>80-100% 合流まで約 3-7 日間細胞を孵化させなさい。2-3 日毎の培養培地を変更します。</li>
	</ol>
</li>
	<li>
KHOS A を分割し、-n 個のセル
	<ol>
<li>フラスコから文化メディアを削除します。</li>
		<li>洗浄 5 mL 滅菌 PBS のセル (1 x) カルシウムとマグネシウム、非付着性のセルまたは分数を削除するなし。</li>
		<li>フラスコ、1 mL 0.05% トリプシン EDTA 溶液を追加し、簡単にトリプシン-EDTA が均等に分散されるようにフラスコを旋回します。</li>
		<li>37 ° C の定温器で 2 〜 3 分または室温で 3-5 分のフラスコを孵化させなさい。セルは切り上げし、切り離されたことを確認する約 400 倍の倍率での顕微鏡の下のセルを確認します。</li>
		<li>セルを取り外したら、一度培地 10 mL を追加し、そっとピペット 10 mL のピペットを使用して単一細胞懸濁液に細胞集塊を破るために数回上下中。</li>
		<li>細胞カウンターを使用してセルをカウントします。2 × 10 の6セルの密度または 1:4 の比率で新しい T75 フラスコに細胞懸濁液をシードします。</li>
		<li>QPI 実験のため播種する前に 80-100% の合流点に成長する細胞を許可します。</li>
	</ol>
</li>
	<li>
KHOS A シードと QPI の - n 個のセル
	<ol>
<li>シード KHOS A とセル カウンター カウント後培地 5 mL を 50,000 300,000 細胞/ウェルの密度で 6 ウェル プレートで - n 個のセル。 注: 播種密度は細胞増殖率およびイメージングの期間に依存するために < 100% 取得、イメージング中に合流。</li>
		<li>添付ファイルを許可するように少なくとも 12 時間のセルを孵化させなさい。</li>
		<li>QPI を行う前に新鮮なメディア媒体を変更します。</li>
	</ol>
</li>
</ol>2. QPI 買収
<ol><li>
カバー スリップを設定
	<ol>
<li>脱イオン水を実行して数回の洗浄によってカバー スリップをきれいにし、カバー スリップを入れ乾燥滅菌層流フード 15 分の浸漬で 75% エタノール溶液で消毒します。</li>
		<li>明白な泡を避けるために 6 ウェル プレート カバー スリップを慎重に配置します。カバー スリップの下が文化メディア (最小 5 mL/ウェル) に没頭していることを確認します。</li>
		<li>(37 ° C、5% CO2、および加湿雰囲気) 少なくとも 15 分平衡にインキュベーターにプレートを配置します。カバー スリップ上に霧を形成、それをきれいに拭くため滅菌綿棒を使用します。</li>
	</ol>
</li>
	<li>
顕微鏡で細胞のイメージング
	<ol>
<li>露出時間、パターン コントラスト、およびホログラム ノイズの自己校正を含むシステムを初期化するソフトウェアを開きます。値が許容される (緑または黄色の範囲で表示) を確認します。</li>
		<li>QPI 顕微鏡のステージ上に 6 ウェル プレートを配置します。</li>
		<li>「Live Capture"をクリックしてし「ソフトウェア フォーカス」で「手動」を選択「顕微鏡の設定」の作業距離を調整することによって細胞の位相画像を粗く集中位相画像のセルのアウトラインを取得しますします。「ソフトウェア フォーカス」で、「自動」に変更します。</li>
		<li>「新しい実験」をクリックして新しいテストを作成します。テンキー、マウスまたは矢印キーを使用してイメージの取得位置の選択を開始します。各選択した後「保存」をクリックします。通常の各サンプルに対して 3-5 場所が選択されます。</li>
		<li>「タイムラプス」セクションのイメージの間隔と期間を設定します。 注: がん細胞を明確に追跡するには、間隔が 5 分 48 時間は組み合わせ QPI 分析の期間として設定より短いでする必要があります。</li>
		<li>「キャプチャ」をクリックして自動的に焦点を当て、設定の時点で画像を取得する実験を開始します。</li>
	</ol>
</li>
</ol>3. データ分析
<ol><li>
細胞の形態解析
	<ol>
<li>「細胞を識別」をクリックバック グラウンド ノイズからのセル領域が分離できるように、「背景のしきい値」の数値の設定を調整します。「オブジェクト サイズ」の設定数を調整それぞれセルに 1 つの核を確認しますします。比較する必要がある最終的な面積と体積の測定に影響を与える可能性がありますこれらのサンプルのための一貫したパラメーターを維持します。手動で必要な場合、「手動で変更、」を使用して、セル セグメントを変更します。</li>
		<li>ソフトウェアの「データの分析」関数を使用して、セル分析。「新分析」をクリックしてして新しいデータ解析をを開始します。セル領域 (2μ m)、光学膜厚 (μ m)、ボリューム (μ3) すべての個々 のセルに対してなど「ソース フレーム」タブと細胞形態のパラメーターに選択した画像をドラッグします。表や図としての散布図のプロットやヒストグラム モードとエクスポート データを選択します。</li>
	</ol>
</li>
	<li>
細胞増殖の解析
	<ol>
<li>(例えば初期、12 h、24 h、36 h、および 48 h) 興味の別の時間ポイントの少なくとも 5 枚の画像を選択します。ソフトウェアによって提供されるレコードの携帯番号。</li>
		<li>倍加時間を推定するには、初期の番号の正規化後細胞数をプロットし、指数関数的成長曲線にフィットします。</li>
	</ol>
</li>
	<li>
細胞運動解析
	<ol>
<li>ソフトウェアの「細胞を追跡する」関数を使用します。「新分析」をクリックして新しいデータ分析を開始します。(例えば、24 時間培養後 4 h)、「ソース フレーム」タブに選択した時間帯のイメージのドラッグ シリーズ ランダムに「セルを追加」選択モード下のセルをクリックしてして 10-30 セルを選択します。エッジとそれらのビューのフィールドの外に移動セルを除外します。</li>
		<li>一連の画像でトラッキングを入念にチェックします。システムはセルのトラックを失うまたは誤ったセルを追跡、こと手動でセルの位置を変更するのには「選択」モードの下で追跡携帯を調整細胞を識別するために「識別する」をクリックするか、「場所の変更」をクリック。</li>
		<li>「プロット運動」におけるセル軌道バラ プロットまたは移行率直さ、移行 (μ m) (μ m) の運動運動速度 (μ m/h) などの「印刷機能」で、そのほかの運動関連パラメーターのプロットを選択します。データを表や図としてエクスポートします。</li>
	</ol>
</li>
</ol>

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利害の対立が宣言されていません。

本研究生体外で、非侵襲的、無料のラベルを用いた QPI ひと骨肉腫細胞の血管新生と血管新生以外の表現型を特徴づける定量的をについて説明します。増殖率、倍加時間、移行の率直さ、運動速度、移行と運動細胞容積、セル厚セル領域を含むこの統合、ハイスループット法による細胞の複数のパラメーターを同時に分析されています。
細胞の形態や細胞の挙動を...

開発・進行固形腫瘍の初期のチェックポイントの 1 つは、血管新生形質、がんの特徴の習得です。この進行には、様々 な生化学的・分子過程1,2,3が含まれます。腫瘍の進行のこの重要なステップにおける技術的な課題は、継続的かつ定量的評価し、公平な方法で生きているがん細胞の血管新生と血管新生以外の表現型を区別するためのツールの欠如です。通常血管新生と非血管新生細胞の細胞の挙動を調査するために使用されている伝統的な試金は高価な試薬・機器を必要とする、たとえば、細胞増殖・遊走アッセイ4,5、 6,7,8,9,10、11,12,13,14か高い専門性と集中を必要とすると同様に相補的な生体内評価4,5,6,8,15,16、時間と労働消費。
最近では、定量位相イメージング (QPI) は、様々 な細胞形態および動作パラメーター17,18,19,の時間経過およびラベリング無料評価を可能にする手法として浮上しています。20,21,22. 従来の光学顕微鏡とは異なり QPI を定量化位相差画素ごとのバリエーション光、光物体を通り抜けるし、変換の光学的厚さとボリューム、自筆を再構築後、直通ができ生きているセル、次の機能の解析: (4) 同時多重パラメーター イメージング (3) ラベル無料画像、(2) 非侵襲とタイムラプス イメージング (1) 定量的イメージング。これらの機能は、QPI に評価し、細胞レベルで病態を理解するための強力なツールを作る。
最近の研究で我々 は定量的評価し、血管新生を区別する QPI を利用 KHOS A および体系的かつ定量的に、細胞形態の分析を組み合わせることでひと骨肉腫細胞の非血管新生 KHOS N の表現型増殖、および運動23。血管新生と血管新生以外のひと骨肉腫細胞の細胞形態および行動のパラメーターを比較した定量的画像解析ソフト、パネルを使用して、5 つの特徴的な違いは、これらの 2 つの識別されました。表現型。この手法は、さまざまな生物学的に関連する細胞の特性を評価するため統合と定量的なプラットフォームを提供します。

<table><tbody><tr><td>T75 flask</td><td>Corning, NY, USA</td><td>353136</td><td></td></tr><tr><td>6-well plates&amp;nbsp;</td><td>Corning, NY, USA</td><td>3506</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Thermo Fisher Scientific, MA, USA</td><td>11965092</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Atlanta Biologicals, GA, USA</td><td>S11550</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermo Fisher Scientific, MA, USA</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific, MA, USA</td><td>10010023</td><td></td></tr><tr><td>Beckman Z1 Coulter counter</td><td>Beckman Coulter, IN, USA</td><td>Z1&amp;nbsp;</td><td></td></tr><tr><td>HoloMonitor M4</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>M4</td><td>Microscope</td></tr><tr><td>Hololid</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>PHI 8020</td><td></td></tr><tr><td>HStudioM4</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>HStudioM4</td><td>Software</td></tr></tbody></table>

a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells

血管新生形質の獲得は腫瘍休眠からの脱出の重要なコンポーネントです。これらのメソッドは時間を調査し、非血管新生と血管新生細胞の表現型を特徴付けるいくつかのクラシックの in vitroアッセイ (例えば増殖、移行、および他の人) と体内のモデルが開発されているが労働集約的なと、多くの場合高価な試薬、機器と専門的な技術を必要とします。最近の研究で血管新生と血管新生以外のひと骨肉腫 KHOS 細胞の時間経過およびラベリング-無料の特徴づけを行う新規定量位相イメージング (QPI) を使用しました。細胞の形態・増殖・運動を含む携帯電話のパラメーターのパネルは、定量的に測定したし、QPI を用いています。この小説と定量的アプローチは、継続的に、非侵襲的シンプルで統一された方法で関連する細胞プロセス、動作、およびがん細胞の特性および他の細胞型を勉強する機会を提供します。このレポートでは、セルの準備、QPI 集録、データ分析など当社の実験プロトコルについて説明します。

図 1は、典型的な細胞形態特性を示しています。画像は、ホログラフィー (図 1 aB) と 2次元画像 (図 1D) として掲載されています。(屈折率と光路長から計算) 光学セルの厚さは線またはセル全体の測定で定量化しました。散布図の面積と厚み KHOS A と図 1E<s...

Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

休止状態とアクティブな癌細胞の表現型は、定量的な位相画像を用いた特徴付けられました。細胞の増殖、移行、および形態の試金を統合し、1 つの単純な方法で分析します。

時間経過、ラベル無料、定量的相休眠とアクティブなひと癌細胞のイメージング研究

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

a-time-lapse-label-free-quantitative-phase-imaging-study-dormant

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

7.4K ビュー.  Boston Children's Hospital. 休止状態とアクティブな癌細胞の表現型は、定量的な位相画像を用いた特徴付けられました。細胞の増殖、移行、および形態の試金を統合し、1 つの単純な方法で分析します。

ビデオ: A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

がん研究

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

侵襲性外科生検に対する脳腫瘍細胞行動の縦腔内イメージング

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

医学

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

ラベル無料三次元定量的な位相画像を用いたリンパ球サブタイプの同定と機械学習

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

免疫・感染学

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

生物学

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

3Dスフェロイドモデルにおける時間分解蛍光イメージングとがん細胞浸潤解析

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

連続イメージングによる細胞死開始の定量化

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

著者スポットライト:過敏反応における細胞死開始を研究するための合理化されたアプローチ 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

デジタル顕微鏡を用いた細胞死開始の研究

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...

時間経過、ラベル無料、定量的相休眠とアクティブなひと癌細胞のイメージング研究

要約

さらに動画を探す

この動画の章

長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために

侵襲性外科生検に対する脳腫瘍細胞行動の縦腔内イメージング

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

ラベル無料三次元定量的な位相画像を用いたリンパ球サブタイプの同定と機械学習

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

3Dスフェロイドモデルにおける時間分解蛍光イメージングとがん細胞浸潤解析

デジタル顕微鏡を用いた細胞死開始の研究

時間経過、ラベル無料、定量的相休眠とアクティブなひと癌細胞のイメージング研究

要約

さらに動画を探す

この動画の章

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

侵襲性外科生検に対する脳腫瘍細胞行動の縦腔内イメージング

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

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