eoe sub articles

EoE Sub Articles

1. Irradiation Set-up
Note: Irradiation of Caco-2 cells was performed at the radiotherapy department of Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) S. Maugeri (Pavia, Italy) with a linear accelerator routinely used to treat different types of cancers.
<ol>
	<li>Set photon X-ray energy to 6 MV peak. The linear accelerator operates in a pulsing regime (time between two consecutive pulses approximately of 4 ms; duration of a single pulse about 5 &#181;s with a dose rate up to 1 &#215; 10-3 Gy/p).</li>
	<li>Place the 6-well plates containing inserts with Caco-2 on the trajectory of X-rays on a 1.4 cm-thick Plexiglas sheet (corresponding to a distance slightly greater than the build-up of the used radiation) at 100 cm from the source of radiation.</li>
	<li>Place a 0.5 cm-thick bolus on each sample before the irradiation occurs to guarantee the backscattered radiation component and the charged particles&#39; equilibrium.</li>
	<li>Irradiate the cells (doses in the range 2 - 10 Gy) with a flat and symmetric (&#177; 2%) 20 x 20 cm2 radiation field and a dose-rate of 3 Gy/min. 
	NOTE: Control &#34;sham&#34;-irradiated cells underwent the same procedural conditions of the irradiated ones, without entering the irradiation room (received dose: 0 Gy). 
	CAUTION: Ionizing radiation-producing devices must be used only by specialized personnel.</li>
</ol>
2. Trans-Epithelial Electrical Resistance (TEER)
<ol>
	<li>Seed 5 &#215; 105 Caco-2 cells one week prior to irradiation in 6-well plate co-culture inserts (polyethylene terephthalate (PET), 2 x 106 pores/cm2).</li>
	<li>1 h before irradiation, measure the TEER with a voltmeter/ohmmeter.</li>
	<li>Irradiate cells as described in steps 1.1 - 1.4.</li>
	<li>Incubate Caco-2 cells in the presence/absence of co-culture with PBMC at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>For the first few hours post-irradiation, measure TEER every hour, and subsequently every 3 hours up to 48 hours.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ThinCert 6 Well Cell Culture Inserts for Multiwell Plates</td>
			<td>Greiner Bio-one</td>
			<td>657610</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Cell Culture Multiwell Plate, 6 Well</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td>Plastic</td>
		</tr>
		<tr>
			<td>Cell Culture Multiwell Plate, 24 Well</td>
			<td>Greiner Bio-one</td>
			<td>662160</td>
			<td>Plastic</td>
		</tr>
		<tr>
			<td>RPMI 1640 without L-Glutamine</td>
			<td>Lonza</td>
			<td>12-167F</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Millicell-ERS voltmeter/ohmeter</td>
			<td>Millipore</td>
			<td>MERS 00001</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

measuring teer in x-ray-irradiated epithelial cells co-cultured with pbmcs

Source: Babini, G., et al. <a href="https://app.jove.com/v/56908">A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC.</a> J. Vis. Exp. (2018)
This video demonstrates the measurement of trans-epithelial electrical resistance in irradiated Caco-2 cells, both in the presence and absence of PBMC cells. This method enables the study of the synergistic effects of radiation and immune cells on the epithelial cell barrier.

Measuring TEER in X-ray-Irradiated Epithelial Cells Co-cultured With PBMCs

1. Irradiation Set-up
Note: Irradiation of Caco-2 cells was performed at the radiotherapy department of Istituto di Ricovero e Cura a Carattere ...

Take a membrane insert containing an X-ray-irradiated epithelial monolayer.
Place the membrane insert into a well containing peripheral blood mononuclear cells, or PBMCs &#8212; immune cells that secrete cytokines, and other signaling molecules.
Place the transepithelial electrical resistance, or TEER electrodes, into the membrane insert.
Apply a small electrical current and measure the resistance to assess monolayer permeability regulated by tight junctions.
Tight junctions are transmembrane proteins linked to the cytoskeleton through scaffold proteins.
Together, these junctional proteins form a robust barrier between cell membranes, restricting the passage of large molecules and pathogens.
Intact junctional proteins impede electrical current flow, resulting in a high TEER, indicating normal monolayer permeability.
X-ray exposure reduces junctional-protein levels, increasing monolayer permeability and lowering TEER.
Moreover, signaling molecules from irradiated cells activate co-cultured&#160; PBMCs that release cytokines, triggering a further reduction in junctional-protein levels and disrupting the barrier integrity.
Consequently, monolayer permeability increases, causing a further decrease in TEER.

measuring-teer-x-ray-irradiated-epithelial-cells-co-cultured-with

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

106 Views. Source: Babini, G., et al. A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC. J. Vis. Exp. (2018) This video demonstrates the measurement of trans-epithelial electrical resistance in irradiated Caco-2 cells, both in the presence and absence of PBMC cells. This method enables the study of the synergistic effects of radiation and immune cells on the epithelial cell barrier. 

Video: Measuring TEER in X-ray-Irradiated Epithelial Cells Co-cultured With PBMCs - Concept

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

JoVE Journal

Chemistry

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Biology

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

Measuring TEER in X-ray-Irradiated Epithelial Cells Co-cultured With PBMCs

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