eoe sub articles

EoE Sub Articles

1. Preparation of reagents and starting materials
<ol>
	<li>Preparation of MDCK cells and sterile cell culture medium
	<ol>
		<li>Cultivate Madin-Darby Canine Kidney (MDCK) cells in modified Eagle&#39;s medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin).</li>
		<li>After thawing, passage MDCK cells at least 2x before infecting them to ensure complete recovery (but less than 30 passages to avoid any drift in cell phenotypes).</li>
		<li>Seed 75 cm2&#160; tissue culture flask(s) containing 30 mL of sterile 1x MEM with 7.5 x 106&#160; MDCK cells and incubate at 37 &#176;C in humidified 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>Preparation of impedance monitoring equipment
	<ol>
		<li>Place the instrument in the incubator at 35 &#176;C and allow it to warm up for at least 2 h.</li>
		<li>Connect it to the control unit placed outside the incubator.</li>
		<li>Proceed to the cleaning procedures as recommended by the manufacturer before starting the remainder of the experiment.</li>
	</ol>
	</li>
	<li>Production of IAV stocks on MDCK cells
	<ol>
		<li>To propagate and amplify H1N1 viruses, seed 7.5 x 106 MDCK cells on two 75 cm2 tissue culture flasks, and incubate for 24 h at 37 &#176;C to reach 90%&#8211;100% confluence.</li>
		<li>Decant the cell culture medium from the cell monolayer in 75 cm2 flasks. Wash the cells with 5 mL of sterile 1x PBS.</li>
		<li>Remove PBS and add 5 mL of 1x PBS to wash the cells again.</li>
		<li>Label one flask as the control and remove PBS before adding 15 mL of virus propagation media (1x MEM with 0% FCS) carefully on the monolayer. Incubate this flask in an incubator maintained at 35 &#176;C with 5% CO2. Use it for comparison after 3 days of propagation.</li>
		<li>Thaw one vial of IAV stock at RT. Dilute the virus to the appropriate concentration in a 1.5 mL tube containing virus propagation media (1x MEM with 0% FCS).</li>
		<li>Remove 1x PBS from the 75 cm2 flask and infect MDCK cells at a multiplicity of infection (MOI) of 1 x 10-3 or 1 x 10-4 plaque-forming units per cell (pfu/ cell) by adding 1 mL of the diluted virus to the cell monolayer.</li>
		<li>Adsorb virus to MDCK cells for 45 min at RT by stirring the flask regularly every 15 min.</li>
		<li>Gently remove the inoculum and add 15 mL of virus propagation media per flask containing 1 &#181;g/mL TPCK-trypsin (trypsin/L-1-tosylamide-2- phenylethyl chloromethyl ketone), to cleave the viral hemagglutinin HA0 into HA1 and HA2 subunits (an event that is required for HA fusion with the endosomal membrane and release of the viral genome).</li>
		<li>Incubate the flasks at 35 &#176;C and 5% CO2 for at least 3 days to replicate the virus.</li>
		<li>Observe MDCK cells under a microscope at 40x magnification and look for cytopathic effects (CPE) on the cells (by comparing them to the cell control flask). If CPE is not complete (i.e., around 80% of the cells are detached from the substrate), put the flasks back in the incubator for an additional 24 h.</li>
		<li>When CPE is complete, decant the cell culture supernatant and centrifuge at 300 x g for 10 min to pellet the cellular debris.</li>
		<li>Transfer the clarified supernatant to a 15 mL tube and aliquot progeny viruses to single-use sterile cryogenic vials. Immediately place the cryotubes at -80 &#176;C to freeze and stock viruses.</li>
	</ol>
	</li>
	<li>Infect MDCK cells with different 10-fold dilutions of a known 6 log10 TCID50/mL titer of H1N1 virus, using reverse pipetting for reproducibility by following the steps below:
	<ol>
		<li>Rinse MDCK cells 2x with 100 &#181;L of MEM with no FCS (virus propagation media). Be aware of removing all media after the second wash to avoid further dilution of the inoculum.</li>
		<li>Add 100 &#181;L of viral suspension in each well using a single-channel pipette. To avoid contamination, proceed by starting from left-to-right then top-to-bottom in the plate while covering the remaining wells with the lid.</li>
		<li>Insert the plate into the cradle pocket of the instrument at 35 &#176;C. Be gentle to avoid sudden movements potentially leading to contamination.</li>
		<li>Start to monitor cell impedance every 15 min during at least 100 h as described in step 2.10.</li>
		<li>After the two cycles of measurements (i.e., 30 min), pause the apparatus by clicking on &#34;Pause&#34; in the &#34;Execute&#34; tab and remove the E-plate from the cradle.</li>
		<li>Add 1 &#181;g/mL TPCK-trypsin to the virus propagation media to cleave the viral hemagglutinin.</li>
		<li>Add 100 &#181;L of virus propagation media containing TPCK-trypsin into each well and insert the E-plate into the cradle pocket. 8. Click &#34;Start/Continue&#34; in the &#34;Execute&#34; tab. 
		NOTE: Do not forget to create a negative control corresponding to mock-infected cells by replacing viral suspension with virus propagation media.</li>
	</ol>
	</li>
</ol>
2. IAV survival kinetics
<ol>
	<li>Expose IAV to saline distilled water at 35 &#176;C and test for their infectivity over time by measuring cell impedance decrease.
	<ol>
		<li>Prepare saline distilled water by adding NaCl to a final concentration of 35 g/L in distilled water. Add 900 &#181;L of saline water into 2 mL cryotubes.</li>
		<li>Add 100 &#181;L of viral stock in saline water and place the cryotubes in an incubator (35 &#176;C, 5% CO2) for 1 h, 24 h, or 48 h.</li>
		<li>Seed 100 &#181;L (containing 3 x 104) of freshly split MDCK cells on a 16-well microtiter plate and grow for 24 h at 37 &#176;C and 5% CO2.</li>
		<li>Infect cells with 100 &#181;L of exposed viruses (previously diluted 10x in culture media) using the reverse pipetting method for reproducibility by repeating section 1.4.</li>
	</ol>
	</li>
	<li>Monitor cell impedance every 15 min for at least 100 h.</li>
</ol>

<table><tbody><tr><td>0.25% Trypsin</td><td>ThermoFisher</td><td>25200056</td><td></td></tr><tr><td>75 cm2 tissue culture flask</td><td>Falcon</td><td>430641U</td><td></td></tr><tr><td>E-Plate 16 (6 plates)</td><td>ACEA Biosciences, Inc</td><td>5469830001</td><td>E-plates are available in different packaging</td></tr><tr><td>FCS</td><td>Life technologies (gibco)</td><td>10270-106</td><td></td></tr><tr><td>MEM 1X</td><td>Life technologies (gibco)</td><td>31095029</td><td></td></tr><tr><td>PBS 1X</td><td>Life technologies (gibco)</td><td>14040091</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life technologies (gibco)</td><td>11548876</td><td></td></tr><tr><td>TPCK-Trypsin</td><td>Worthington</td><td>LS003740</td><td></td></tr><tr><td>xCELLigence Real-Time Cell Analysis Instrument S16</td><td>ACEA Biosciences, Inc</td><td>380601310</td><td>The xCELLigence RTCA S16 instruments are available in different formats (16-well, 96-well, single or multi-plate</td></tr></tbody></table>

cellular impedance-based survival kinetics assay to assess virus survival

Source: Labadie, T. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021).
This video describes a cellular impedance-based technique to study the survival kinetics of viruses that induce cytopathic effects. The method measures the electrical impedance of virus-infected adherent cells pre-treated with saline.

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival

Source: Labadie, T. et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021).
This video describes ...

Viruses bind to the host-cell receptor and internalize into an endosome. The endosomal membrane fuses with the viral membrane, releasing viral contents inside the cell.
Upon reaching the nucleus, the viral genome transcribes into mRNA and later translates to form viral proteins. The viral components enter the cytoplasm and assemble into virions, which pinch out of the cell and cause cytopathic effects &#8213; changes in the host cell&#39;s morphology, and death.
To assess virus survival kinetics by measuring host-cell impedance, take a multiwell plate coated with impedance-measuring microelectrodes at its bottom. Add a host-cell suspension to the wells, and incubate to allow the cells to adhere to the electrode surface, and grow.
Next, take saline pre-treated viral suspensions with different viral loads. Saline treatment supplies the virus with chloride, which produces hypochlorous acid that kills it &#8213; reducing the viral infectivity. Add these viral suspensions into their respective wells.
Measure the cellular impedance &#8213; a phenomenon of opposing the flow of electric current through the electrodes &#8213; by the adherent cells.
Viruses infect adherent host cells and change their morphology, causing the detachment of the morphed cells from the electrodes. Over time, as more cells get infected, the host cell-mediated cellular impedance decreases.
Plot the cell index, which is the change in impedance due to infected cell detachment, over time.

cellular-impedance-based-survival-kinetics-assay-to-assess-virus

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

126 Aufrufe. Quelle: Labadie, T. et al., Überwachung des Überlebens von Influenzaviren außerhalb des Wirts mittels Echtzeit-Zellanalyse. J. vis. exp. (2021). Dieses Video beschreibt eine zelluläre Impedanz-basierte Technik, um die Überlebenskinetik von Viren zu untersuchen, die zytopathische Effekte induzieren. Die Methode misst die elektrische Impedanz von virusinfizierten adhärenten Zellen, die mit Kochsalzlösung vorbehandelt wurden. 

Serie: Zellulärer impedanzbasierter Überlebenskinetik-Assay zur Beurteilung des Überlebens von Viren - Konzept

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Impedanzbasierte Echtzeitmessung der Krebszellmigration und -invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Forschung

JoVE Journal

Krebsforschung

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Beurteilung von Eierstockkrebs Sphäroid Bindung und Invasion von Mesothelzellen in Echtzeit

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Medizin

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival

Transkript

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival