1。細胞計数の重要性<ol><li>カウント細胞はin vitroでの細胞培養でのために必要ですが面倒なステップです。 </li><li>一貫性のある細胞濃度は、実験的な再現性と精度を確保する。 </li><li>細胞数は、その後の実験に、トランスフェクションまたは感染症のために細胞を播種し、セルベースアッセイの準備を、不死化または変形の評価、細胞の健康と増殖率を監視するために重要である。 </li><li>細胞計数のスピードと正確さの必要性にもかかわらず、、400研究者の71％は血球計算盤を使用して、カウント細胞を調査した。血球計算法は安価ですが、それは面倒で不正確なカウントで、その結果ユーザーのバイアスや誤用、対象となります。 </li><li>血球計算法の誤差の源は次のとおりサンプルの不均一な細胞分布、多すぎたり少なすぎたり、細胞のサンプルで、特定のセルが定義されている計数の領域内に収まるかどうかの主観的な意思決定、血球計数器の汚染、ユーザからユーザへの変化を、および血球計充填率の変化。 </li><li>手動の計数に関連付けられて退屈を軽減するために、研究者の29％が自動化された細胞計数装置を用いて細胞を数える。しかし、ほとんどの研究者のために、これらの大型ベンチトップ機器に関連付けられている価格は、それらの使用の障害となります。 </li><li>セプターの細胞計数は、比較的低コストでカウントコールターの自動化と精度を提供する自動化されたハンドヘルドデバイスです。 </li><li>システムは、センシング、信号処理、データストレージ、およびグラフィカルな表示、アナログとデジタルハードウェアの組み合わせを使用して小型化した形式でインピーダンスベースの粒子検出のコールター原理を採用しています。 </li><li>使い捨てのセンサーは、細胞の大きさとサブミクロンおよびサブピコリットルの分解能での細胞容積による差別を可能にする微細加工、細胞感知ゾーンに設計されています。 </li><li>精密液体処理チャンネルと電子機器で拡張された、セプターの細胞計数は、グラフィカルにヒストグラムとして細胞集団の統計情報が表示されます。 </li></ol> 2。カウントのための単一細胞懸濁液の調製<ol><li>セプターのセルカウンタを使用するには、細胞の単一の細胞懸濁液を希釈することにより開始する、リン酸塩で緩衝生理食塩水。 mL当たり10,000-500,000細胞の濃度が最適です。 </li><li> 1.5mlマイクロチューブに希釈した細胞100μLを移す。 </li></ol> 3。セプターを使用して実行するセルのカウント<ol><li>計器の背面にあるトグルを押し続けると、王笏のセルカウンタをオンにして、表示​​される画面上の指示を待ちます。 </li><li>プロンプトが表示されたら、機器の前面に向かって対向する電極センサーパネルでセプターユニットの端にセンサーを取り付けます。 </li><li>セプターのセンサーは、電子検出ゾーンと統合された細胞検知電極と、精密成形サンプリングチャンバーで構成されています。センサーにより、サブピコリットルの分解能でサブミクロンの解像度でセルサイズを区別し、細胞の体積になります。 </li><li>一貫性のあるセンサ技術は、同じサンプル量がカウントされると正確なサイズ測定が行われることを保証します。 </li><li>一度センサーが取り付けられている、次のように画面上の指示に従ってカウント処理の各ステップを実行します。 </li><li>画面が読み込むときに&quot;開始するためにプランジャーを押しながら、&quot;プランジャーを押し下げる。画面にはその後、&quot;水没センサーを。&quot;と表示されます細胞懸濁液にセンサーとセンサーに細胞懸濁液を吸引するためにプランジャーを離して水没。 </li><li>プランジ​​ャーは、電気信号​​として機能し、センサーにサンプルに加え、デッドボリュームが描画されます。それは、ピペッティングと同様プランジングのスピードや正確さは関係ありません。このプロセスには数秒かかります。準備ができたら、楽器音が鳴ります、および&quot;ロードされたサンプルは、&quot;画面に表示されます。 </li><li>サンプルをセンサーに読み込まれると、それは、マイクロチャネル通過し、細胞のサイズとセルの体積を評価するための感知ゾーンを通って描画されます。サンプル50μLがカウントされるとセンサーがカウントを停止するために楽器を伝えます。 </li><li>カウントが完了した後、測定器の画面は&quot;完全なカウント。センサーを削除し、破棄してください。&quot;と表示されますプロンプトに従い、センサーを取り外して廃棄します。 </li><li>楽器は、その画面上のセルの大きさや直径のヒストグラムだけでなく、mL当たりの細胞濃度が表示されます。 </li></ol> 4。データ解析<ol><li>カウントが完了するとヒストグラムが計器に表示された後、トグルボタンを押してゲートを適用します。 &quot;自動ゲーティング&quot;を選択するかのどちらかを自動的にヒストグラムプロファイルに基づいてゲートを設定する、または前回データからのゲートを使用するために&quot;最後に使用してください&quot;。 </li><li>に関係なく、選択の、ゲートが手動でデータを微調整するために移動することができます。左のゲートを選択してスクロールし、もう一度トグルボタンを押してください必要に応じてゲートを移動するggle。 </li><li>その後、右側のゲートを活性化し、右側のゲートを配置するトグルをスクロールするには、再度トグルを押してください。もう一度クリックすると、新しい細胞の濃度、平均細胞容積と直径が表示されます。 </li><li>トグルは、日付、時間、平均セルサイズ、および細胞の容積を意味する間、表示を切り替えるにはどちらの側に移動することができます。 </li><li>最大72ヒストグラムに楽器自体に格納することができます。パーソナルコンピュータへデータをアップロードするには、表示画面上にUSBケーブルをポート経由で測定器を取り付けます。 </li><li> PC上で、二重&quot;接続&quot;をクリックしてソフトウェアを起動するにはセプターのソフトウェアアプリケーションをクリックしてください。すべてのカウントは自動的にソフトに移植する。 </li><li>セプターのアプリケーションソフトウェアは、ボリュームまたは直径のいずれかで文化の人口分布を詳細にヒストグラムが表示されます。ダウンロードしたファイルは、生データが指定されたセルの直径またはセルのボリュームのビンに表示されるMicrosoft Excelでは、にエクスポートすることができます。 </li></ol> 5。代表的な結果<ol><li>ナインティーンの異なる細胞株をカウントし、理論的な出発濃度に調製したコールターカウンターを用いて決定された。これらの細胞懸濁液は、さらに理論的な細胞の濃度で細胞懸濁液を得るために希釈した。 </li><li>セプターの細胞計数は、細胞濃度を決定するために使用されていました。図1は、理論的な細胞濃度と一緒に5代表的な細胞株について測定された5希釈を示しています。 </li><li> R2の値によって示されるように直線性の高いは、そのセプターのカウントが広い線形動作範囲にわたって、テストされた細胞株のための信頼性の高い方法であるを示しています。 </li><li>セプターのカウントの全体的な相対精度は、セプターのカウントから得られたデータは、コールターのカウントから得られたカウントと比較されている図2に示されている。 </li><li>セプターの細胞数のログは、コールターのカウントのログに対してプロットし、テストしたすべての細胞株を越え、セプターの数はコールター数に一致する、を示しています。 </li><li> 19細胞株のカウントがZ2コールターカウンター、セプターのセルカウンター、Viのセルまたは無数のシステム、およびhemocytemeterなどの自動視覚ベースのカウンタを用いて行った。 </li><li>カウントは、サスペンションと同一の希釈液を開始し、同じセルを使用して、メーカーの指示に従って行った。図3に示す結果は、セプターで測定された細胞の濃度が密接に高い直線性を持つ理論的な細胞濃度に一致数えることを示している。 </li><li> ％の変動係数、またはCVは、それぞれのカウントシステムのための各細胞株のそれぞれの希釈を計算した。図4は、細胞濃度とカウント方法に関しては、19細胞株の平均％のCVを示す。 </li><li>動作範囲内で、セプターの細胞計数は小さく、標準偏差と変動の小さい平均係数を表示し、視覚ベースの血球計計数よりも正確です。 </li><li>セプターのカウントは10倍の血球計算法よりも速く、あまりにも他の自動化されたカウンタよりも高速です。図5、SF9、MCF7、およびHEK293細胞ではこのビデオで説明する方法を用いてカウントした。 </li><li>細胞濃度を測定するために必要な平均時間を記録した。血球計算法は、サンプルごとに一度だけ実行されたことに注意してください。 </li><li>セプターは、セルサイズに基づいて測定を行います。したがって、細胞形態の変更については、ヒストグラムに取り込むことができます。健全な文化は、不健康な細胞をしながら、素敵なベル形のガウス分布を表示し溶解、そして左にヒストグラム（図6、左パネル）のシフトを引き起こす。 </li><li>同様に、50％の死細胞の人口が50％、生細胞と混合した場合、ヒストグラムのシフトも（図6、右のパネル）が観察された。 </li></ol><img src="/files/ftp_upload/2204/2204fig1.jpg" alt="図1" /><img src="/files/ftp_upload/2204/2204fig2.jpg" alt="図2" /><img src="/files/ftp_upload/2204/2204fig3.jpg" alt="図3" /><img src="/files/ftp_upload/2204/2204fig4.jpg" alt="図4" /><img src="/files/ftp_upload/2204/2204fig5.jpg" alt="図5" /><img src="/files/ftp_upload/2204/2204fig6.jpg" alt="図6" /><img src="/files/ftp_upload/2204/2204fig7.jpg" alt="図7" />

<ol>
	<li>Barghshoon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Counting Survey. , (2009).</li><li>Tucker, K. G., Chalder, S., al-Rubeai, M., Thomas, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hybridoma+cell+number,+viability,+and+morphology+using+fully+automated+image+analysis.">Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.</a> Enzyme Microb Technol. 16 (1), 29-35 (1994).</li><li>Houwen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fifty years of hematology innovation: the Coulter principle. , (2003).</li></ol>

キャスリーンOngena、C​​handreyeeダス、ジャネットL.スミス、ソニアギル、そしてグレースジョンストンは、この資料に使用される楽器を生成するミリポア、によって雇用されている。

他のカウントの方法からの結果にセプターのセルカウンタのパフォーマンスを比較すると、我々はこの新しい携帯、自動細胞計数器は、広い動作範囲にわたって正確に、速く、信頼性の高い細胞数を提供すると結論付けている。セプターのカウントの機能は、センサー先端部とコールター原理に基づく洗練されたカウントの計測に埋め込まれた精密エンジニアリング技術の結果です。パフォーマンスの質は速度を上げるとセルベースアッセイの再現性を向上させる方法としてカウントセプターを示唆している。ハンドヘルドの形式は、細胞培養のフードで迅速な退屈フリーカウント権利を許可します。

<table border="1"><tbody><tr><td> 材料名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> 40μmの王笏センサと笏2.0ハンドヘルド自動化セルカウンター</td><td> EMDミリポア</td><td> PHCC20040 </td><td></td></tr><tr><td> 60μmのセプターのセンサと笏2.0ハンドヘルド自動化セルカウンター</td><td> EMDミリポア</td><td> PHCC20060 </td><td></td></tr><tr><td>セプターセンサ、60μmの</td><td> EMDミリポア</td><td> PHCC60050 </td><td>センサーの50パック</td></tr><tr><td>セプターセンサ、40μmの</td><td> EMDミリポア</td><td> PHCC40050 </td><td>センサーの50パック</td></tr><tr><td>ユニバーサル電源アダプタ</td><td> EMDミリポア</td><td> PHCCPOWER </td><td> </td></tr><tr><td>笏O -リングキット</td><td> EMDミリポア</td><td> PHCCOCLIP </td><td> Oリング2個、一つのフィルターカバー</td></tr></tbody></table>

determining cell number during cell culture using the scepter cell counter

細胞のカウントは、多くの場合、in vitroでの細胞培養のために必要であるが、退屈なステップです。一貫性のある細胞濃度は、実験的な再現性と精度を確保する。細胞数は、その後の実験に、トランスフェクションまたは感染症のために細胞を播種し、セルベースアッセイの準備を、不死化または変形の評価、細胞の健康と増殖率を監視するために重要である。細胞数は、特に細胞応答の定量的測定のために、、正確で一貫性のある、かつ早くなることが重要です。 細胞計数のスピードと正確さのためのこの必要性にもかかわらず、400の研究者の71％は血球計算盤を用いて細胞をカウント1を調査した。血球計算法は安価ですが、それは面倒で不正確なカウントで、その結果ユーザーのバイアスや誤用、対象となります。 Hemocytometersは、細胞懸濁液は、指定されたボリュームにロードされ、顕微鏡下でカウントされている特殊な光学ガラスで作られています。血球計算法の誤差の源は、次のとおりサンプルの不均一な細胞分布、多すぎたり少なすぎたり、細胞のサンプルで、特定のセルが定義されている計数の領域内に収まるかどうかの主観的な意思決定、血球計数器の汚染、ユーザからユーザへの変化を、および血球計充填率2の変化。 手動の計数に関連付けられて退屈を軽減するために、研究者の29％がデバイスを数える自動化された細胞を用いて細胞を数えるが、これらは視覚ベースのカウンタ、コールター原理を用いて細胞を検出するシステム、またはフローサイトメトリー1が含まれます。ほとんどの研究者のための、自動化システムを使用する主な障壁は、これらの大規模なベンチトップ測定器1に関連する価格です。 セプターの細胞計数は、比較的低コストでカウントコールターの自動化と精度を提供する自動化されたハンドヘルドデバイスです。システムは、コールターの検出のためにアナログとデジタルハードウェアの組み合わせを使用して小型化した形式のインピーダンスベースのパーティクルの検出3の原理、信号処理、データストレージ、およびグラフィカル表示を採用しています。使い捨てチップは、セルサイズとサブミクロンおよびサブピコリットルの分解能での細胞容積による差別を可能にする微細加工、細胞感知ゾーンに設計されています。精密液体処理チャンネルと電子機器で拡張された、セプターの細胞計数は、グラフィカルにヒストグラムとして表示される細胞集団の統計を報告します。

Watch this Scientific Journal Video about Determining Cell Number During Cell Culture using the Scepter Cell Counter at JoVE.com

Determining Cell Number During Cell Culture using the Scepter Cell Counter

セプターのセルカウンターは、セルを数えるセルの直径とボリュームを監視し、一つの文化から次の細胞集団の健康と品質を確認するために使用するために使用できる携帯型自動化されたデバイスです。

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 ...

determining-cell-number-during-cell-culture-using-scepter-cell

Research

JoVE Journal

Biology

37.8K ビュー.  Millipore Inc. セプターのセルカウンターは、セルを数えるセルの直径とボリュームを監視し、一つの文化から次の細胞集団の健康と品質を確認するために使用するために使用できる携帯型自動化されたデバイスです。

ビデオ: Determining Cell Number During Cell Culture using the Scepter Cell Counter

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

冷凍細胞から出発培養：ヒトES細胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

生物学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Moxi Zミニ自動セルカウンターを用いた哺乳類細胞数、細胞サイズと細胞の健康の決定

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

胚発生時に細胞運命のコミットメントをテストするために割球植

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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.

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 ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.