eoe sub articles

EoE Sub Articles

1. Single-cell Sampling
<ol>
	<li>Micromanipulation 
	&#8203;NOTE: To allow downstream applications like whole-genome analysis (WGA), isolated single cells will be added to 2 &#181;L of cell lysis master mix. Prepare a master mix according to the number of cells to be processed. Calculate extra volumes for loss due to pipetting and place cell lysis master mix on ice.
	<ol>
		<li>Prepare cell lysis master mix (components of WGA kit,&#160;Table of Materials and Reagents). Briefly, mix 2.0 &#181;L of reaction buffer, 1.3 &#181;L of octylphenoxypolyethoxyethanol (10% solution), 1.3 &#181;L of 4-(1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol (10% solution), 2.6 &#181;L of a proteinase K solution, and 12.8 &#181;L RNase/DNase-free water to obtain a master mix for 10 samples (total volume 20 &#181;L). 
		NOTE: For more samples, increase the volumes accordingly. &#160;&#160;&#160;&#160;&#160;</li>
		<li>Use a grease pen to draw an area holding the cell suspension in place. Adjust area and volume if the cell density is too high (i.e., mark a larger area on the glass slide, load 1x PBS and an aliquot of the cell suspension).</li>
		<li>Pipette the entire target cell suspension onto the glass slide.</li>
		<li>Transfer the glass slide to a microscope equipped with a micromanipulator. Let the cells settle for 5 min.</li>
		<li>Prepare 0.2 mL PCR tubes loaded with 2.0 &#181;L of cell lysis master mix (prepared in step 1.1.1.) and store it on ice.</li>
		<li>Collect single cells in 1 &#181;L of 1x PBS using the micromanipulator and transfer the cells into a tube containing cell lysis master mix. 
		NOTE: For transferring micromanipulated cells into the cell lysis buffer, place the capillary directly into the 2 &#181;L of lysis solution and eject until bubbles can be seen.</li>
		<li>Briefly spin down the sample at 2000 x&#160;g&#160;for 3 s using a desktop microfuge to collect all liquid at the bottom of the tube. Place the samples on ice.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Phosphate Buffered Saline (1x PBS)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10010-015</td><td></td></tr><tr><td>Vacutainer EDTA (or Na-heparin) tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>366450 (or 366480)</td><td>Used as reaction tube for ex vivo capture of target cells</td></tr><tr><td>Pasteur pipettes 150 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Volac</td><td>D810</td><td>Used for the low-volume application of cells to the C&amp;amp;R</td></tr><tr><td>Grease pen&amp;nbsp;&amp;nbsp;</td><td>Dako</td><td>S2002</td><td>Used on glass slides to hold cell suspension in place</td></tr><tr><td>Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario&amp;nbsp;&amp;nbsp;</td><td>Zeiss/Eppendorf</td><td></td><td>Use micromanipulator at hand</td></tr><tr><td>Microcapillaries (25 &amp;micro;m in diameter), CustomTip Type I&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Eppendorf</td><td>930001201</td><td>Used for micromanipulating cells</td></tr><tr><td>0.2 mL PCR tubes&amp;nbsp;</td><td>Biozym</td><td>710920</td><td></td></tr></tbody></table>

single cell sampling using micromanipulation: a technique to isolate single cells from cancer cell suspension

Source: Chen S. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
This video describes the technique for isolating single cells from a pool of target cells to carry out downstream analysis. In this method, a micromanipulator fitted to a microscope is used for high-precision isolation of a single target cell.

Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)
This ...

Single-cell sampling enables isolating a single live cell from a suspension of cancer cells for downstream analysis.
Begin the isolation with a cancer cell suspension. Transfer the desired volume of this suspension onto a glass slide. Leave the slide undisturbed for a while to reduce particle movement and allow the cells to settle.
Now, transfer the slide under a microscope equipped with a micromanipulator connected to a microcapillary tip. This high-precision instrument facilitates physical interaction with cells in the sample at a microscopic level.
Direct the microcapillary tip close to a single target cell. By applying suction, collect the target cell into the microcapillary. Now, transfer the microcapillary directly into a tube containing the desired collection buffer.
Eject the microcapillary contents to release the captured single cell into the buffer. Store the isolated live single cell on ice until further analysis.

single-cell-sampling-using-micromanipulation-technique-to-isolate

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Assays and techniques

1.2K Views. Fonte: Chen S. et al. Pre-arricchimento delle cellule bersaglio e amplificazione dell'intero genoma per la caratterizzazione a valle di singole cellule. J. Vis. Exp. (2018) Questo video descrive la tecnica per isolare singole cellule da un pool di cellule bersaglio per eseguire analisi a valle. In questo metodo, un micromanipolatore montato su un microscopio viene utilizzato per l'isolamento ad alta precisione di una singola cellula bersaglio. 

Video: Campionamento di singole cellule mediante micromanipolazione: una tecnica per isolare singole cellule dalla sospensione di cellule tumorali - Concept

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Micromanipolazione delle cellule tumorali circolanti per l'analisi molecolare a valle e la valutazione del potenziale metastatico

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Ricerca

JoVE Journal

Ricerca sul cancro

Micromanipulation of Chromosomes in Insect Spermatocytes

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell.
The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.

In this protocol, we describe the selection and preparation of appropriate cells for micromanipulation and the use of a piezoelectric micromanipulator to reposition chromosomes within those cells.

Micromanipolazione dei cromosomi in insetto spermatociti

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and ...

Biologia dello sviluppo

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

Singola cella multisala trascrizione inversa Polymerase Chain Reaction dopo Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension

Trascrizione

Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension