Name: 视频: 功能化基于线的靶细胞分离:一种从加标外周血中分离癌细胞的离体技术 - 概念
Uploaded: 2023-04-30T13:28:58.000+00:00
Duration: 1 min 49 s
Description: 1.1K 次观看. 来源:Chen S. et al. 用于单细胞下游表征的靶细胞预富集和全基因组扩增。J. Vis. Exp. （2018 年） 在本视频中，我们演示了从外周血样本中分离表达 EpCAM 的上皮癌细胞。该技术涉及使用经过 EpCAM 抗体功能化的医用级金属丝来捕获靶细胞。然后将捕获的细胞提取到缓冲溶液中。

eoe sub articles

EoE Sub Articles

NOTE: This protocol describes the isolation of HT-29 cells (human colon cancer cell line) from PBS or from artificial mixtures of HT-29 cells and peripheral blood. The same experiment was performed with two additional cell lines (LNCaP and VCaP) and can theoretically be performed with all cells expressing EpCAM.
1. Preparation of Target Cells
<ol>
	<li>Cell culture and labeling of cells 
	NOTE: In this protocol, cells are cultured in 75 cm&#178; culture flasks. Please adjust the amounts of reagents accordingly if other cell culture devices are used (e.g.,&#160;25 cm&#178; culture dishes, 6-well plates,&#160;etc.). All liquids used are pre-warmed to 37 &#176;C in a water bath. If not indicated otherwise, all protocol steps are performed at room temperature (RT).
	<ol>
		<li>Culture HT-29 cells in McCoy&#39;s 5a Modified Medium supplemented with 2 mM L-glutamine, 20 mM Hepes, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin at 37 &#176;C in a 5% CO2&#160;atmosphere.</li>
		<li>When cells are 90% confluent, remove the medium and rinse the cells with 10 mL of 1x PBS.</li>
		<li>To harvest cells, add 2 mL of the cell detachment solution (Table of Materials and Reagents) to the cells and incubate the cells for approximately 5 min at 37 &#176;C. 
		NOTE: The detachment solution contains proteolytic and collagenolytic enzymes in 1x PBS, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and 3 mg/L phenol red. Reduce the pre-warming time of the cell detachment solution to a minimum as this will be inactive after 1 h at 37 &#176;C. Cover the whole cell layer to obtain optimal cell detachment.</li>
		<li>Check the detachment of cells using an inverted microscope.</li>
		<li>Transfer all detached cells to a 50 mL tube prefilled with 10 mL of cell culture medium (same as used in step 1.1.1.) and resuspend the cells by pipetting.</li>
		<li>Place the remaining cell suspension on a horizontal roller mixer at RT and proceed to the labeling step.</li>
	</ol>
	</li>
	<li>Live cell labeling of target cells
	<ol>
		<li>Dilute 1 &#181;L of 5 mM CFSE (supplied in dimethyl sulfoxide, DMSO) in 1 mL of 1x PBS pre-warmed at 37 &#176;C to obtain the 5 &#181;M ready-to-use CFSE labeling solution. 
		NOTE: For optimal staining results, use freshly prepared solutions.</li>
		<li>Prepare a ready-to-use DNA staining solution (Table of Materials and Reagents) in 1x PBS and store it at 2-6 &#176;C protected from light. 
		NOTE: For optimal staining results, use freshly prepared solutions.</li>
		<li>Get the cells from the horizontal roller mixer (step 1.1.6.) and pellet the cells at 300 x&#160;g&#160;for 10 min. Remove the supernatant and resuspend the cells in 10 mL of 1x PBS.</li>
		<li>Rinse the cells again with 1x PBS and resuspend the cell pellet in a 500 &#181;L ready-to-use CFSE labeling solution.</li>
		<li>Incubate the cells at 37 &#176;C for 15 min and collect the cells after centrifugation at 300 x&#160;g&#160;for 3 min.</li>
		<li>Resuspend the labeled cells in 1 mL of pre-warmed cell culture medium and allow the cells to regenerate at 37 &#176;C for 30 min.</li>
		<li>Harvest the cells by centrifugation at 300 x&#160;g&#160;for 3 min and resuspend the cell pellets in 1 mL of ready-to-use DNA staining solution at 37 &#176;C for 10 min.</li>
		<li>Pellet the cells, remove the supernatant, and resuspend the cells in 4 mL of 1x PBS. Assess the cell density using a hemocytometer and check for fluorescence labeling using a fluorescence microscope equipped with 4&#39;,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) filters (Figure 1A&#160;and 1E).</li>
		<li>Centrifuge the cells at 300 x&#160;g&#160;for 3 min and resuspend the cell pellet according to the cell densities needed for the respective experiment as given in the next section and place on ice.</li>
	</ol>
	</li>
</ol>
2. Charging the Wire
NOTE: Cells can be isolated from target cell/peripheral blood spikings at the milliliter scale or by providing a low number of cells at a microliter scale. Whereas the former approach allows for mimicking rare-cell conditions in peripheral blood, the latter may be the preferred way to attach a few cells for the purpose of protocol optimization/testing.
<ol>
	<li>Charging the wire using a large volume of cell suspensions
	<ol>
		<li>Prepare a 2% BSA blocking solution by dissolving 0.8 g of BSA in 40 mL of 1x PBS (cell culture use). Dissolve BSA by initial vortexing and subsequent incubation at RT on a horizontal roller mixer for 10-20 min.</li>
		<li>Rinse empty EDTA tubes/sodium heparin tubes with 2 mL of 2% BSA to remove anticoagulant residues and discard the solution. Block the tube surface by incubation at RT with 5 mL of 2% BSA on a horizontal roller mixer at 15 rpm for 30 min and discard the solution.</li>
		<li>Dilute labeled cells (section 1.2.) to a density of 100,000 cells/mL and use 5 mL to charge the wire.</li>
		<li>Add 5 mL of the labeled cell suspension (500,000 cells in total) to the tube. Avoid bubbles when adding the cell suspension.</li>
		<li>Remove the wire from the storage compartment as well as the rubber cap holding the wire and rinse it in 1x PBS (Figure 2).</li>
		<li>Penetrate the rubber tube cap of the EDTA tube (step 2.1.4.) with the help of a syringe needle (20 G) and insert the wire such that the functional part is fully immersed in the cell suspension when the cap is back on the tube and the tube placed on the tilted roller mixer. 
		NOTE: The triple-helical functional part of the wire should be covered with cell suspension throughout the charging.</li>
		<li>Rotate the tubes on a tilted roller mixer at 5 rpm for 30 min to allow the cells to attach to the wire.</li>
		<li>Rinse the wire with 5 mL 1x PBS and store it in the dark at 2-6 &#176;C in a 15 mL tube containing 1x PBS until visual examination. 
		NOTE: The functional part of the wire must always be submerged in PBS to avoid harming the cells.</li>
	</ol>
	</li>
	<li>Isolating target cells from artificial mixtures with peripheral blood&#160;(alternative charging method to: 2.1. Charging the wire using a large volume of cell suspensions)&#160;
	<ol>
		<li>Dilute labeled cells (section 1.2.) to obtain cell suspensions with high cell density (&#62; 1,000,000 cells/mL), thereby keeping the volume of cell suspension added to the peripheral blood low.</li>
		<li>Add 500 to 500,000 cells to 5 mL of peripheral blood and mix them by inverting the tube.</li>
		<li>Remove the wire from the storage compartment, remove the rubber cap holding the wire and rinse it in 1x PBS.</li>
		<li>Penetrate a new tube cap with the non-functional part of the device using a syringe needle (20 G) such that the functional part is fully immersed in the spiked blood when the cap is back on the tube and the tube is placed on the tilted roller mixer. 
		NOTE: The triple-helical functional part of the wire should be covered with cell suspension throughout the charging.</li>
		<li>Incubate the tube on the tilted roller mixer at 5 rpm for 30 min at RT.</li>
		<li>Rinse the wire three times in 1x PBS and store it in the dark in a 15 mL tube containing 1x PBS until visual examination. 
		NOTE: The functional part of the wire must always be submerged to avoid harming the cells.</li>
	</ol>
	</li>
	<li>Charging the wire from low-volume cell suspensions&#160;(alternative charging method to: 2.1. Charging the wire using a large volume of cell suspensions)
	<ol>
		<li>Resuspend the labelled cells at 1,000,000 cells/mL in 1x PBS.</li>
		<li>Fix a 150 mm disposable glass Pasteur pipette horizontally on a rack.</li>
		<li>Remove the wire from the storage compartment but keep the yellow rubber cap holding the wire.</li>
		<li>Place the wire in the pipette such that the functionalized part is situated in the tip of the Pasteur pipette not touching the glass. 
		NOTE: The tip of the wire should not stick out of the Pasteur pipette tip. Avoid plugging the rear end of the Pasteur pipette tip with the rubber cap holding the wire. If attached tight, cell suspensions cannot be loaded from the other side into the pipette tip.</li>
		<li>Load 15 &#181;L of the cell suspension into the tip of the Pasteur pipette covering the functionalized part of the wire.</li>
		<li>Incubate the wire for 10 min. Manually quarter-turn the assembled wire/Pasteur pipette tip every minute.</li>
		<li>Remove the wire from the Pasteur pipette tip, rinse it in 5 mL of 1x PBS and store it in the dark at 2-6 &#176;C in a 15 mL tube containing 1x PBS until visual examination. 
		NOTE: The functional part must always be submerged in 1x PBS to avoid harming the cells.</li>
	</ol>
	</li>
</ol>
3. Counting Cells on the Wire
NOTE: Always keep the functional part of the wire submerged in 1x PBS to avoid harming the cells.
<ol>
	<li>Use a grease pen to draw a rectangle area on a glass slide and add 500 &#181;L of 1x PBS.</li>
	<li>Bend the non-functional part of the wire and place it on the glass slide such that the functional part is immersed in 1x PBS. 
	NOTE: Fit the area of the rectangle and volume of 1x PBS to completely cover the functional part of the wire. Use a double-faced adhesive tape to mount the non-functional part of the wire on the slide.</li>
	<li>Visually inspect both sides of the wire to enumerate the captured cells (Figure 1B&#160;and 1F). 
	NOTE: The presence of cells is determined using a fluorescence microscope equipped with filters for DAPI and FITC. Both sides of the wire can be inspected and the number of cells either assessed (for high cell numbers) or counted (only feasible if low numbers of cells are attached to the wire). This step can be skipped if the enumeration of cells is not necessary.</li>
	<li>After scanning, place the wire back into the 15 mL tube containing the 1x PBS, store the wire in the dark. 
	NOTE: Most of the non-functional part of the wire can be cut (including the bend) and removed easing storage.</li>
</ol>
4. Detachment and Recovery of Target Cells
NOTE: Detachment of the cells shall be done within 4 h after isolation.
<ol>
	<li>Dissolve 4 mg of the release buffer component (Table of Materials and Reagents) per mL of 1x PBS and filter the solution through a 0.2 &#181;m sterile filter to obtain the ready-to-use buffer. 
	NOTE: The polymer of the wire is composed of a hydrogel which is functionalized with anti-EpCAM antibodies allowing binding to EpCAM-presenting target cells. The release buffer contains a carbon-oxygen proteolytic enzyme capable of cleaving the hydrogel. Proteolytic cleavage results in degradation of the polymer and thus detachment of captured cells.</li>
	<li>Pre-warm the release buffer at 37 &#176;C for 5 min.</li>
	<li>Add 1.6 mL of release buffer to fill a 1.5 mL reaction tube. 
	NOTE: Tubes designed for 1.5 mL reaction volumes allow application of 1.6 mL which is necessary for submerging the functional part of the wire.</li>
	<li>Incubate the functional part of the wire in the release buffer at 37 &#176;C (water bath) for 20 min. Use the rubber cap shipped with the wire instead of the tube cap to fix the wire in the 1.5 mL tube to avoid contamination during incubation in the water bath.</li>
	<li>Transfer the wire/tube assembly to a shaker at RT and 500 rpm for 15 min. 
	NOTE: The shaker has an orbit of 1.5 mm.</li>
	<li>Place the wire/tube assembly in a centrifuge and spin it at 300 x&#160;g&#160;for 10 min.</li>
	<li>Remove the wire from the tube, close the cap and centrifuge the tube again at 300 x&#160;g&#160;for 10 min. 
	NOTE: The wire can be stored in 1x PBS at 2-6&#160;&#176;C in the dark for cell counting to assess detachment efficiency/check for cells remaining on the wire.</li>
	<li>To reduce the volume of the cell suspension, remove all but 100 &#181;L (micromanipulation) or alternatively 300 &#181;L (cytocentrifugation and subsequent laser microdissection) of the supernatant and immediately proceed to single-cell sampling.</li>
</ol>

<table><tbody><tr><td>Gilupi Release Buffer</td><td>Gilupi</td><td>GIL083</td><td>Used to detach cells from the wire</td></tr><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>Foetal 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>Vacutainer EDTA (or Na-heparin) tubes</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>Detektor CANCER03 DC03</td><td>Gilupi</td><td>GIL003</td><td>Functionalized wire capable of isolating and detaching EpCAM-positive 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>Sterile syringe filter (0.2 &amp;micro;m)</td><td>Corning</td><td>431219</td><td>For filtering freshly prepared Gilupi Release Buffer</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>Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario</td><td>Zeiss/Eppendorf</td><td></td><td>Use micromanipulation at hand</td></tr><tr><td>Microcapillaries (25 &amp;micro;m in diameter), CustomTip Type I</td><td>Eppendorf</td><td>930001201</td><td>Used for micromanipulating cells</td></tr><tr><td>0.2 mL PCR tubes</td><td>Biozym</td><td>710920</td><td></td></tr><tr><td>Cytocentrifuge and equippment</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>MembraneSlide 1.0 PET</td><td>Zeiss</td><td>415101-4401-050</td><td>Membrane-coated glass slides used for laser microdissection</td></tr></tbody></table>

functionalized wire-based target cell isolation: an ex vivo technique to isolate cancer cells from spiked peripheral blood

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)
In this video, we demonstrate the isolation of EpCAM-expressing epithelial cancer cells from peripheral blood samples. The technique involves the use of a medical-grade wire functionalized with EpCAM&#160;antibodies to capture the target cells. The captured cells are then subsequently extracted into a buffer solution.

<img alt="Figure 1" src="/files/ftp_upload/20395/20395_Figure1.jpg" /> 
Figure 1: Visualizing labeled cells.&#160;(A,E)&#160;Cells before charging: Cells are labeled with CFSE and counterstained with a DNA intercalating dye showing a range of CFSE (green) signal intensity.&#160;(B,F)&#160;Labeled cells captured on the wire.&#160;(C,G)&#160;Wire after cell-detachment ...

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

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

Epithelial cell adhesion molecules, or EpCAMs, are surface transmembrane glycoproteins overexpressed in rapidly proliferating epithelial cells in prostate cancers. To isolate EpCAM-expressing cells, begin with a medical-grade wire bearing a functionalized gold-coated tip. Functionalization layers a polymeric gel containing covalently bound EpCAM-specific antibodies.
Immerse the wire into a peripheral blood suspension containing fluorescently labeled cancer cells. Ensure that the functionalized part remains dipped in the blood suspension. The EpCAMs on the cancer cells recognize and bind to their specific antibodies functionalized on the wire.
Wash with a buffer to remove any unbound cells from the wire surface. Bend the wire and mount it on a buffer-containing glass slide, with the functionalized part immersed in the buffer. Use a fluorescence microscope to check bright fluorescence from cell cytoplasm and nuclei and confirm the presence of captured cells on the wire.
Treat the wire with a release buffer. The proteolytic enzymes in the buffer cleave the polymeric gel, releasing the antibody-cancer cell complexes into the buffer. Centrifuge the wire assembly to collect the released antibody-cell complexes in the pellet. Remove the wire and the enzyme-containing supernatant. Resuspend the target cell-containing pellet in a suitable medium.

functionalized-wire-based-target-cell-isolation-an-ex-vivo-technique

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Assays and techniques

1.1K 次观看. 来源:Chen S. et al. 用于单细胞下游表征的靶细胞预富集和全基因组扩增。J. Vis. Exp. （2018 年） 在本视频中，我们演示了从外周血样本中分离表达 EpCAM 的上皮癌细胞。该技术涉及使用经过 EpCAM 抗体功能化的医用级金属丝来捕获靶细胞。然后将捕获的细胞提取到缓冲溶液中。 

视频: 功能化基于线的靶细胞分离:一种从加标外周血中分离癌细胞的离体技术 - 概念

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

科研

JoVE Journal

癌症研究

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

用数字聚合酶链反应检测循环核糖核酸 ROS1 和 RET 融合记录的血液检测方法

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

医学

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

成績單

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood