Name: Video: Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population - Concept
Uploaded: 2023-04-30T13:29:15.000+00:00
Duration: 1 min 39 s
Description: 945 Views. Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018) This video describes the technique for obtaining a single cell from a heterogeneous cell population using laser capture microdissection. The single isolated cell can be used for further genomic studies or cancer research.

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1. Single-cell Sampling
<ol>
	<li>Laser Microdissection 
	&#8203;NOTE: Single cells will be added to 4.5 &#181;L of cell lysis master mix (components of WGA kit, Table of Materials and Reagents) for WGA.
	<ol>
		<li>Prepare the cell lysis master mix according to Czy&#380; et al. by mixing 5.0 &#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), resp., 2.6 &#181;L of a proteinase K solution and 34.8 &#181;L of RNase/DNase-free water to obtain a master mix for 10 samples (total volume 45 &#181;L). Place the master mix on ice. 
		NOTE: For more samples, increase the volumes accordingly.</li>
		<li>UV-treat the membrane-coated slides suited for laser microdissection. Place the slides in a DNA crosslinker device and expose them to the highest UV for about 10 min.</li>
		<li>Assemble the membrane-coated slide in a cytocentrifuge and cytospin the entire cell suspension at 300 x g for 5 min. 
		NOTE: When using an in-liquid system, where the cells are cytocentrifuge onto the slide in solution, remove the supernatant after cytocentrifugation and apply a dry-spin at 1140 x g for 1 min.</li>
		<li>Directly proceed to laser microdissection or let the cytospins air-dry overnight and freeze the samples at -20 &#176;C for long-term storage.</li>
		<li>Pipette 4.5 &#181;L of cell lysis master mix into the cap of a 0.2 mL PCR tube and harvest single cells by means of laser microdissection.</li>
		<li>Retrieve the PCR tube from the laser microdissection microscope and close the tube. Collect all liquid at the bottom of the tube by short-spin and place the sample on ice.</li>
		<li>Proceed with single-cell WGA or store the isolated samples at -80 &#176;C for up to 1 month before further processing.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>McCoy&#039;s 5A Medium (1x) suppl. with L-Glutamine</td><td>Thermo Fisher Scientific</td><td>16600-082</td><td>Culture medium used for HT-29 cells, adapt culture medium to cell line used for the experiments</td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>PAA</td><td>S11-001</td><td>Supplement for McCoy&#039;s 5A Medium</td></tr><tr><td>Fetal Bovine Serum Gold</td><td>PAA</td><td>A15-151</td><td>Supplement for McCoy&#039;s 5a Modified Medium</td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Biowest</td><td>L0018-100</td><td>Supplement for McCoy&#039;s 5a Modified Medium</td></tr><tr><td>Phosphate Buffered Saline (1x PBS)</td><td>Thermo Fisher Scientific</td><td>10010-015</td><td></td></tr><tr><td>Accutase</td><td>Biowest</td><td>L0950-100</td><td>Cell detachment solution (cell culture)</td></tr><tr><td>Water bath</td><td>GFL</td><td>Type 1003</td><td>Used for prewarming solutions</td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific</td><td></td><td>Used to incubate cells (cell culture)</td></tr><tr><td>50 mL reaction tubes</td><td>VWR</td><td>525-0403</td><td></td></tr><tr><td>Roller mixer</td><td>Stuart Scientific</td><td>SRT6D</td><td>Used to attach cells to the wire while keeping the cells from sedimenting</td></tr><tr><td>CellTrace CFSE Cell Proliferation Kit</td><td>Thermo Fisher Scientific</td><td>C34554</td><td>Used to label living cells (cytoplasmic label)</td></tr><tr><td>Hoechst 33342</td><td>H3570</td><td>Thermo Fisher Scientific</td><td>Used to stain DNA (nucleic counterstain)</td></tr><tr><td>Centrifuge (equipped for holding 15 mL and 50 mL reaction tubes)</td><td>Thermo Fisher Scientific</td><td>Heraeus Megafuge 40R</td><td>Used for pelleting cells</td></tr><tr><td>Observer Z1 (Fluorescence/laser microdissection microscope)</td><td>Carl Zeiss Microimaging</td><td></td><td>Inverted (immunofluorescence) microscope capable of laser microdissection; Fluorescence microscopes must be able to detect Hoechst 33342 (DAPI filter) and CFSE (FITC filter)</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4503-50G</td><td>Used for coating glass/plastic surfaces</td></tr><tr><td>MS1 Minishaker</td><td>Sigma-Aldrich</td><td>Z404039</td><td>Used for vortexing</td></tr><tr><td>Detektor CANCER03 DC03</td><td>Gilupi</td><td>GIL003</td><td>Functionalized wire capable of isolating and detaching EpCAMpositive cells (also referred to as catch&amp;amp;release, C&amp;amp;R)</td></tr><tr><td>Syringe needles (G20)</td><td>VWR</td><td>613-0554</td><td>Used to puncture rubber caps in order to allow the non-functional part of the C&amp;amp;R to lead through it</td></tr><tr><td>Neubauer chamber</td><td>Roth</td><td>T729.1</td><td>Used to count cells</td></tr><tr><td>Pasteur pipettes 150 mm</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</td><td>Dako</td><td>S2002</td><td>Used on glass slides to hold cell suspension in place</td></tr><tr><td>Delfia PlateShaker</td><td>Perkin Elmer</td><td>1296-003</td><td>Used for agitation during cell detachment</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td>30,125,150</td><td></td></tr><tr><td>Ampli1 WGA Kit</td><td>Silicon Biosystems</td><td></td><td>To be ordered via Silicon Biosystems</td></tr><tr><td>0.2 mL PCR tubes</td><td>Biozym</td><td>710920</td><td></td></tr><tr><td>Cytocentrifuge and equipment</td><td>Hettich</td><td>Universal 32</td><td>Use cytocentrifuge at hand</td></tr><tr><td>Thermo cycler</td><td>Bio-Rad</td><td>DNA Engine Dyad</td><td>Every thermo cycler with heated lid can be used</td></tr><tr><td>Microcentrifuge</td><td>Roth</td><td>CX73.1</td><td>Use desk-top centrifuge at hand</td></tr><tr><td>UV Stratalinker 1800</td><td>Stratagene</td><td>400072</td><td>Use DNA cross linker at hand</td></tr></tbody></table>

single cell sampling using laser capture microdissection: a technique to harvest target cells from a heterogeneous cell population

Source: Chen S. et al. <a href="https://www.jove.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 obtaining a single cell from a heterogeneous cell population using laser capture microdissection. The single isolated cell can be used for further genomic studies or cancer research.

Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population

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

Laser capture microdissection helps to procure a single target cell from a cell mixture prepared on a glass slide. Begin by taking a suitable glass slide coated with a UV-permeable, biochemically inert membrane on its surface. Expose the slide to UV light to render it more hydrophilic for better cell adherence.
Assemble the slide into a cytocentrifuge apparatus.&#160; Add cell suspension onto the funnel port of the apparatus. Cytocentrifuge to allow the centrifugal force to concentrate and deposit a monolayer of cells on a small area on the glass slide. Allow the slide to air-dry.
Place the slide, sample side up, under a laser microdissection microscope. Define the region surrounding the cell of interest. Now, focus the laser beam to dissect the target-cell-containing region along with the membrane coating. Next, defocus the laser beam and pulse it to generate pressure. This energy catapults the microdissected region into the inverted cap of a pre-assembled collection tube containing a buffer placed above the slide.
Retrieve the tube and close its cap. Spin the tube to bring the buffer containing the single target cell to the tube bottom. Refrigerate the tube until further analysis.

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945 Views. Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018) This video describes the technique for obtaining a single cell from a heterogeneous cell population using laser capture microdissection. The single isolated cell can be used for further genomic studies or cancer research. 

Video: Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population - Concept

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

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Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

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RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population

Transcript

Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones