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

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

For micromanipulation, transfer the entire 100 microliters of cell suspension onto a glass slide. Then, place the glass slide on a microscope equipped with a micromanipulator and allow the cells to settle for 5 minutes. Then, in 0.2-milliliter PCR tubes, pipet 2 microliters of cell lysis master mix and transfer on ice. Use the micromanipulator to collect a single cell in 1 microliter of 1X phosphate-buffered saline.
Then, transfer the cell directly into 2 microliters of cell lysis master mix. Use a desktop microfuge to quickly spin down the samples at 2000 x g for 3 seconds to collect the liquid at the bottom of the tube.

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Universidad de Cartagena UDEC

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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>

Source: Chen S. et al. <a href="https://jove.unicartagenaproxy.elogim.com/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.

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.

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

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