Akshata Anchan was funded by the Neurological Foundation of New Zealand for the Gillespie Scholarship (grant reference: 1628-GS) and First Fellowship (grant reference: 2021 FFE). The research cost was also partially funded by the Neurological Foundation Fellowship-2021 FFE and the University of Auckland Faculty Research Development Fund. James Hucklesby was funded by a scholarship from the Auckland Medical Research Foundation. Thanks to the Baguley team and Auckland Cancer Society Research Centre for the patient-derived New Zealand Melanoma NZM cell lines.

1. Using ECIS to monitor changes in brain endothelial barrier integrity in response to various treatments
<ol>
	<li>Setting up the software, array station, and machine
	<ol>
		<li>Connect the 96-well array station to the machine. Place the array station in a plastic bag and place it in the incubator at least 1 h before the assay to allow it to warm up. Remove the plastic bag immediately before the experiment.</li>
		<li>Once ready, turn on the machine and open the corresponding software on the computer. Press the Setup button under the Collect Data tab. The software will complete a connection check, which will detect the type of adapter attached (e.g., 96-well array station) and display red wells in the depicted plate map. This states that the software detects the adapter but cannot sense a plate attached. The system is now ready for the 96-well plate.</li>
	</ol>
	</li>
	<li>Preparing the 96-well biosensor plate 
	NOTE: The 96-well plate arrangement is used. The base of each well is patterned with interdigitating fingers of gold-plated electrodes. The plates come in sterile packs that should only be opened in a sterile hood/biological safety cabinet. The gold electrodes must first be chemically stabilized by adding 10 mM cysteine to maintain the electrode capacitance.
	<ol>
		<li>Prepare the 10 mM cysteine in a sterile trough and using a multi-channel, carefully pipette 100 &#181;L into each well. Incubate the cysteine for 15 min at room temperature and then carefully aspirate, pipetting at the edges of the well only, to avoid scratching the electrode. Wash the cysteine off at least 2x with sterile deionized water (not PBS) using the same pipetting technique. 
		NOTE: The plate electrodes can also be stabilized electrically using warm media on the ECIS apparatus, instead of using cysteine coating, as detailed in the manufacturer&#39;s protocol.</li>
		<li>Prepare ECM substrate to act as a basement membrane on the bottom of the well. 
		NOTE: ECM substrates like collagen, or laminin may be chosen depending on the requirement of the brain endothelial cell line. Here we use collagen for a human cerebral microvascular endothelial cell line (hCMVEC).</li>
		<li>Prepare 1 &#181;g/cm2 rat-tail collagen I dissolved in 0.02 M acetic acid in a fresh, sterile trough (100 &#181;L per well). Leave the collagen for 1 h at low light in the sterile hood, then wash 2x carefully with deionized water and careful pipetting. 
		NOTE: Ensure that as much of the deionized water is removed as carefully as possible after the last wash and let the plate dry in the hood. It is recommended to use the plate soon after coating; however, it can be stored in water at 4 &#176;C temporarily (under a week). The plate is now ready for use.</li>
	</ol>
	</li>
	<li>Preparing the brain endothelial cells for seeding onto the 96-well biosensor plate 
	NOTE: It is recommended to first optimize the growth phase and seeding density of the endothelial cell line to be used. The hCMVEC line was used in this protocol, which takes 48 h to reach confluency after seeding at 20,000 cells per well in a 96-well plate.
	<ol>
		<li>Harvest the brain endothelial cells in a sterile hood using a gentle dissociation reagent and conduct an accurate cell count. Use 20,000 hCMVECs in 100 &#181;L of media per well for a steady growth phase for 48 h. Prepare excess brain endothelial cells to ensure optimal pipetting of all 96 wells. 
		NOTE: Hence, for a 96-well plate, approximately 2,000,000 cells in 10 mL of prewarmed media are needed.</li>
		<li>Transfer the brain endothelial cells to a fresh sterile trough and using a multi-channel pipette, carefully add 100 &#181;L (20,000 cells) of the cell suspension per well. Resuspend the cells gently in the trough regularly to avoid the endothelial cells settling over time. Ensure 1 well has media only and no cells to allow mathematical modeling (cell-free well) and that the entire plate is seeded within 30 s.</li>
	</ol>
	</li>
	<li>Starting the experiment
	<ol>
		<li>Carefully attach the plate to the adapter in the incubator by moving the red clips on either side of the plate adapter outwards. Align the A1 well of the plate with the A1 region of the adaptor. Gently hold the plate in place with one hand on top and click the red clips back in place with the other hand. 
		NOTE: Ensure that the plate is stable in the adaptor.</li>
		<li>Close the incubator and press Setup again. Look for green wells in the plate map indicating correctly detected wells. Any wells showing red have a connection error. To fix this, re-attach the plate quickly, 
		NOTE: Excessive time spent trying to fix wells with poor connections may compromise cell health.</li>
		<li>Press Check to re-check the attachments as absolute impedance readouts. This will take several minutes.</li>
		<li>Press the arrow under the plate map to ensure the correct plate catalog is selected. In this case, it is 96w20idf.</li>
		<li>Press the multifrequency button to ensure impedance is measured at multiple AC frequencies to allow future mathematical modeling.</li>
		<li>Press Start to begin the experiment. Allow the cells to proliferate for ~48 h and form their typical monolayer of high resistance, as measured by an increase in ohms.</li>
	</ol>
	</li>
	<li>Treating the brain endothelial cells with cancer cells or cytokines on the 96-well plate 
	NOTE: As the 96-well plate allows concurrent testing of multiple stimuli, prepare a clear plate map with desired treatment options (Supplemental Figure S1-bottom). Each treatment can and must be conducted at least in triplicate (three replicate wells) to allow statistical analyses; however, four replicates are shown in Supplemental Figure S1.
	<ol>
		<li>At 48 h, check the growth of the brain endothelial cells. Ensure a plateau is reached before preparing treatment solutions. 
		NOTE: Different endothelial cell lines may take different lengths of time to reach a plateau. Hence, their growth phase and seeding density must be optimized prior to conducting this experiment. Wells should typically plateau within the same hour. If not, they might be compromised or pipetted inaccurately and must not be included.</li>
		<li>For treatment with cytokines, prepare appropriate concentrations of the cytokines in prewarmed complete media along with their relevant vehicle and media controls.
		<ol>
			<li>Prepare the cytokines at 2x the final desired concentration as they will be diluted 1:1 when added to each well containing brain endothelial cells.</li>
			<li>To allow each treatment to be conducted in triplicate, prepare all cytokines in over 300 &#181;L of total volume (i.e., at least 350 &#181;L total), for treatment addition of 100 &#181;L per well.</li>
		</ol>
		</li>
		<li>For treatment with cells, harvest the cells (three different patient-derived melanoma cell lines used in this protocol) with a gentle dissociation reagent and conduct an accurate cell count. Perform cell-based treatments at top effector:target ratios (E:T ratios) of 1:1 where the treatment cells are the effector cells (e.g., melanoma cells) and the brain endothelial cells are the target cells i.e. add 20,000 melanoma cells to 20,000 brain endothelial cells per well.
		<ol>
			<li>Serially dilute for smaller E:T ratios of 1:10 or 1:100 where required. To allow treatment in triplicate wells, count the cells and prepare them in over 300 &#181;L of total volume (i.e., at least 350 &#181;L total) for treatment addition of 100 &#181;L per well.</li>
		</ol>
		</li>
		<li>Label 1.1 mL polypropylene cluster tubes, which come in 8-tube strip format (i.e., 1 mL strip tubes), according to the 96-well plate map (Supplemental Figure S1-Top). Insert the strip tubes in the strip tube plates and autoclave them before the experiment. Add the total volume of treatment cytokines or cells (i.e.&#160;full 350 &#181;L of treatment) to the sterile strip tubes and leave them in the incubator to keep warm.</li>
		<li>On the software, press Pause to pause the experiment. This may take a few seconds to complete. Once paused, open the incubator and carefully unclip both red clips with one hand while stabilizing the plate with the other hand. Keep the experiment paused and bring the plate to the sterile hood.</li>
		<li>Remove the treatment strip tubes from the incubator. Using a multichannel pipette, carefully resuspend the contents of the treatment strip tubes and then add 100 &#181;L of treatment from the strip tubes to the correct wells on the 96-well plate without touching the bottom of the well. Carefully add treatment to all wells and media only to the cell-free well and complete this step in under 5 min for a full 96-well plate, as excessive cooling will affect the resistance of the endothelial monolayer.</li>
		<li>Return the treated plate to the machine, attach it as done previously, and then check the electrode connections to the adaptor again by pressing Setup | Check.</li>
		<li>Ensure that the correct plate catalog and multifrequency acquisition settings are selected, and press Resume to resume the experiment. Allow the experiment to progress and run it until the desired endpoint.</li>
		<li>Press Finish to complete the experiment, which automatically saves the recorded abp. file. Navigate to File | Export Data to retrieve this file as a xls. or csv. file for further analyses.</li>
	</ol>
	</li>
</ol>
2. Using cellZscope to monitor changes in brain endothelial barrier integrity in response to various treatments
<ol>
	<li>Preparing the apparatus&#39; metal components, adaptor, and porous membrane insert
	<ol>
		<li>Clean the metal components sequentially with deionized water, then 70% ethanol, and then deionized water again.</li>
		<li>Autoclave the bottom electrode or &#34;pots&#34; and top dipping electrodes and sterilize all other components of the Cell Module with 70% ethanol.</li>
		<li>In a sterile hood, install all of the 24 bottom electrodes/&#34;pots&#34; by screwing them into the Cell Module, taking care to maintain sterility throughout.</li>
		<li>Add 900 &#181;L of base media to the wells. 
		NOTE: Base media is added to the bottom well, which acts as the basal compartment of the endothelium. It is recommended to not use serum in this compartment to more reliably replicate the basal environment of the brain endothelium.</li>
		<li>Connect the dipping electrodes magnetically to the Cell Module lid and close the lid over the wells so the dipping electrodes slot into the bottom electrode pots.</li>
		<li>Attach the Cell Module to the adaptor in an incubator and leave it for at least 1 h to equilibrate. At this stage, open the software to load the experimental plate map under the Layout tab, thereby embedding the treatment plate map into the files, which will ultimately contain the data.</li>
		<li>Where coating is required, coat the membrane inserts in a sterile hood by using a sterile 24-well culture plate to hold the inserts and adding the ECM substrate (e.g., collagen) to the inner section of the well, as detailed in section 1.2.3 Use a single-channel pipette to load the ECM substrate into each well carefully.</li>
	</ol>
	</li>
	<li>Preparing the brain endothelial cells for seeding onto the membrane insert and starting the experiment
	<ol>
		<li>Harvest and count the brain endothelial cells as per section 1.3, but prepare&#160;the final required cell titration in a 50 mL centrifuge tube. Ensure the harvested cells are in warm complete media, including all required growth factors and serum.</li>
		<li>Using a single-channel pipette, carefully seed the brain endothelial cells (80,000 in this protocol) into the apical chamber of a well insert in 350 &#181;L of complete media.</li>
		<li>Include a cell-free well by pipetting complete media only into one of the inserts.</li>
		<li>Remove the Cell Module from the incubator and place it in the sterile hood. Transfer the prepared inserts into the bottom electrode pots using a pair of tweezers to handle only the upper supports of the insert. Avoid bubbles forming underneath the membrane.</li>
		<li>Place the Cell Module back in the incubator over the adapter.</li>
		<li>In the software, in the experiment tab, under the Spectrum section, select Start at 1 Hz and Stop at 100 kHz to acquire data over the entire frequency range; select fine steps.</li>
		<li>Under Wait time, select 15 min to permit continuous measurements at the fastest rate conducted by the software.</li>
		<li>Press Start to begin the experiment and allow brain endothelial cells to proliferate and form a monolayer with high resistance. This step takes approximately 48 h.</li>
	</ol>
	</li>
	<li>Treating the brain endothelial cells with cancer cells and cytokines on the membrane inserts
	<ol>
		<li>At 48 h, prepare the treatment cytokines or cells (e.g., melanoma cells) as per section 1.5, including conducting cell counts where applicable, and collect in 1 mL strip tubes or equivalent.</li>
		<li>Prepare treatment at correct concentrations for adding 70 &#181;L to each well (insert). Place treatments in the incubator to keep warm.</li>
		<li>As the instrument herein conducts measurements every 15 min, choose a treatment time that begins immediately after a measurement is completed. At this time, remove the Cell Module from the incubator and gently wipe it with 70% ethanol using lint-free wipes.</li>
		<li>In a sterile hood, open the Cell Module and carefully pipette 70 &#181;L of the treatment into the apical chamber of the respective insert. Do this using a single channel pipette and add in a circular motion, avoiding bubbles. Complete this step in under 10 min to return the Cell Module to the adapter before the next measurement starts.</li>
		<li>Press Stop to end the experiment. Export the data directly to a xls. file under the File | Export Tab. Name and save this file appropriately after opening it.</li>
	</ol>
	</li>
</ol>

Impedance-Based Biosensors in Investigating Brain Endothelial Barrier Strength

The authors have no conflicts of interest to declare.

Therapeutic studies on diseases that affect the BBB must consider the importance of brain endothelial barrier integrity and regulation. For example, brain endothelial barrier disruption is critically investigated in the metastasis of cancer to the brain from other anatomical sites. This is because the brain endothelium forms the first barrier against circulating tumor cells. As mentioned earlier in the introduction, in vitro studies on endothelial barrier integrity need to be reproducible, non-invasive, label-fr...

This article discusses current trends in the assessment of microvascular cells. We specifically detail the use of two commercially available platforms for measuring the barrier properties of cerebral microvascular endothelial cells. Endothelial cells are blood-facing cells, which line the vessel wall. However, cerebral microvessels are unique as they help form the protective blood-brain barrier (BBB)1,2,3. The BBB functions to regulate the transport of molecules from the blood to the brain. Peripheral diseases that affect the central nervous system (CNS) are commonly linked to a functional failure of the BBB4,5. The anatomical structures that form the BBB are not present at the blood-tissue interface elsewhere in the body6. These anatomical structures comprise pericytes, which are located close to brain endothelial cells, and regulate their proliferation and permeability; astrocytic foot processes, which are involved in nutrient shuffling and anatomical support7,8; microglia, which are the resident macrophages in the brain, often implicated in neuroinflammation and ischaemia9,10,11,12, and the brain endothelium, which forms a monolayer of tightly adhered cells without fenestrations13,14. The brain endothelium is typically known as the &#39;brain endothelial barrier&#39; and forms a structural and functional barrier in five distinct ways. First, the paracellular barrier component is formed by adhesion at lateral cell-cell junctions. Second, the transcellular barrier component is sustained by regulating endocytosis. Third, a specialized basement membrane anchors and supports the endothelium via a rich extracellular matrix comprising largely of collagen15,16. The last two mechanisms are through enzymes and transporters that help regulate the metabolism of drugs and uptake of large molecules, respectively17.
The paracellular interactions form the major component of the brain endothelial barrier, facilitated by tight junctions (TJs), comprising membrane proteins claudins, occludin, and junctional adhesion molecules (JAMs)18. Strong homotypic binding of the membrane proteins forms the first structural barrier, though JAMs also link to accessory proteins zonula occludens, thus linking the TJs to the actin cytoskeleton19,20. The actin links place the TJs in the apical region of the endothelium21, which functionally polarizes the endothelial cells to form the structural barrier on this apical or &#34;blood-facing&#34; side. In the basolateral region of the endothelium, highly specialized adherens junctions (AJs) play a regulatory role in maintaining cell morphology. AJs comprise calcium-dependent cadherins, which link the cytoskeleton of neighboring endothelial cells through the catenin family complex20,22. Vascular endothelial cadherin (VE-Cadherin) is one such cadherin, which regulates the expression of TJ proteins and overall endothelial barrier function to maintain endothelial monolayer integrity23,24,25,26,27. Inflammatory modulators, such as tumor necrosis factor-alfa (TNF&#945;), signal VE-cadherin internalization away from cell junctions, leading to destabilization of the endothelial barrier28,29,30. Platelet endothelial cell adhesion molecule (PECAM)31 is another AJ cadherin that stabilizes and remodels endothelial junctions32,33. The density of these junctional proteins restricts the flow of electrons through the paracellular space between endothelial cells. This attribute is utilized to measure endothelial barrier strength or trans-endothelial electrical resistance (TER) across confluent cell monolayers like the brain endothelium.
Therapeutic studies focus on the brain endothelium due to its vital role at the blood-brain interface. Several diseases negatively affect the brain endothelium, including neuroinflammatory conditions like multiple sclerosis, stroke, neurodegenerative diseases, and cancer34,35,36,37. Once the brain endothelial barrier is disrupted, disease progression significantly worsens as the brain is effectively exposed to the harmful stimuli in the blood38. We have previously shown that inflammatory mediators and metastatic melanoma cells disrupt the brain endothelial barrier by using two technologies that measure endothelial barrier strength39,40,41.
Electric cell-substrate impedance sensing (ECIS) is an impedance-based biosensor that permits real-time and label-free assessment of endothelial cell barrier integrity. Herein, assay wells are lined with gold-plated electrodes, which introduces alternating current (AC) to the assay system. Brain endothelial cells are seeded into these wells, which means that AC can be applied through the cells. (Figure 1A-well; side view). This establishes the electrical circuit, which is used to calculate the impedance (Figure 1A-circuit diagram). The impedance increases when the brain endothelial cells adhere to the plate and begin forming their paracellular junctions. The impedance plateaus when the endothelial cells become confluent, forming a monolayer, and restricting current flow. Application of AC at different frequencies influences the route of flow of current through the endothelial cells. Current flows through the endothelial cell body when applied at a higher frequency (&#62;104 Hz). This provides information on the capacitance of the cell monolayer, used to assess cell attachment and spreading. At low frequencies (102-104 Hz) the membrane impedance is high, restricting current flow through the cells. In this case, the majority of the current navigates between the cells. At approximately 4,000 Hz, the resistance to current flow is attributed mostly to the endothelial cell-cell junctions, via the intercellular space.Therefore, any change in resistance at this frequency provides information regarding endothelial barrier integrity.
Whilst raw impedance measurements can provide insight into barrier properties, the ECIS software can then mathematically model the total resistance measured across multiple AC frequencies and more precisely, separate it into two key parameters of barrier integrity. These parameters are the paracellular resistance between the lateral membranes of neighboring cells (resistance beta-Rb; paracellular barrier; Figure 1A-green arrows), and the basolateral resistance between the basal cell layer and the electrode (resistance alpha-Alpha; basolateral barrier; Figure 1A-blue arrows). A third modeled parameter is also measured as the cell membrane capacitance (Cm; Figure 1A-red arrows). The Cm displays the capacitive flow of current through the cells, indicative of cell membrane composition. Herein, changes in the Rb or paracellular barrier indicate alterations in the TJs and AJs, crucial in maintaining endothelial barrier integrity. To reliably interpret the Rb, four key assumptions are made, as developed by Giaever and Keese42 and critically discussed by Stolwijk et al.43. Although these assumptions are important for ensuring the validity of ECIS modeling, they are readily met by a confluent endothelial monolayer.
Like ECIS, the cellZscope permits the measurement of changes in endothelial barrier resistance; however, the cells are cultured on a porous membrane insert. In this system, the electrical circuit is between two electrodes on either side of a membrane insert. The endothelial monolayer is cultured on top of this membrane insert, allowing for measurement of trans-endothelial electrical resistance (TER) (Figure 1B-well; side view). As with ECIS, in this system, the total impedance can be attributed to several barrier components, dependent on the frequency of current applied44. At low frequencies, electrode capacitance (CEl) dominates the total impedance of the system. Alternatively, at high frequencies, the resistance of the media (Rmedium) dominates the total impedance. Hence, the most useful measurements fall within the midfrequency range (102-104 Hz), which provides information regarding two key components of the endothelial barrier (Figure 1B-circuit diagram). First, at 103-104 Hz, the cell layer capacitance (CCl) dominates the overall impedance as the membrane resistance (Rmembrane) is high enough to be neglected, and current flows predominantly across the capacitor. Hence, the CCl indicates changes to resistance through the cell membrane. Alternatively, TER predominantly imparts the overall impedance at 102-103 Hz, where current flow is channeled through junctional spaces between neighboring cells, held together by junctional proteins. Hence, this provides information on the paracellular component of the endothelial barrier, as seen previously with Rb on ECIS.
Figure 1C details how specific regions of the brain endothelium are disrupted by&#160;treatment with&#160;melanoma cells. This disruption is detected by the biosensors by a change in the flow of current through the paracellular space (measured as Rb or TER); the basolateral space (measured as Alpha); and the cell membrane (measured as Cm or CCl). We used both biosensors detailed in this introduction to measure brain endothelial barrier change following treatment with various stimuli such as cytokines or invasive melanoma cells. The measured resistance decreases if a given stimulus disrupts the endothelial barrier, creating a path of least resistance to allow current flow. Hence, a decrease in &#34;barrier resistance&#34; suggests loss of barrier integrity or brain endothelial barrier disruption. In these assays, we have studied this disruption by interpreting resistance and modeled parameters in real time. The application of ECIS and cellZscope in addressing such research questions are detailed elsewhere39,41,45,46.
In vitro research allows the discovery of crucial disease mechanisms by revealing molecules and functional pathways, which progress the disease. However, this requires reliable replication of the disease in vitro, which substantially differs from a functioning body. In an ideal scenario, in vitro research should be reproducible, non-invasive, label-free, quantitative, and mimic structural influences found in vivo. In this article, we detail the methodology for using these two contending technologies to measure treatment-induced changes in brain endothelial barrier integrity. We discuss the advantages of combining their results to provide a more comprehensive picture of barrier disruption and share limitations that still need to be overcome.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>aMEM</td>
			<td>Gibco</td>
			<td>12561072</td>
			<td>Melanoma cell base media</td>
		</tr>
		<tr>
			<td>cellZscope array</td>
			<td>nanoAnalytics</td>
			<td>cellZscope2; software v4.3.1</td>
			<td>TER measuring biosensor array</td>
		</tr>
		<tr>
			<td>Collagen I&#8212;rat tail</td>
			<td>Gibco</td>
			<td>A1048301</td>
			<td>ECM substrate for coating</td>
		</tr>
		<tr>
			<td>dibutyryl-cAMP</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>ECIS array</td>
			<td>&#160;Applied Biophysics</td>
			<td>ECIS Z&#920;; software v1.2.163.0</td>
			<td>Rb/Alpha measuring biosensor array</td>
		</tr>
		<tr>
			<td>ECIS plate</td>
			<td>&#160;Applied Biophysics</td>
			<td>96W20idf&#160;</td>
			<td>96-well biosensor plate</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>12203C-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>305050-061</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>hCMVEC</td>
			<td>Applied Biological Materials</td>
			<td>T0259</td>
			<td>Brain endothelial cell line</td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>PeproTech</td>
			<td>PTAF10015100</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H-3393</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>hFGF</td>
			<td>PeproTech</td>
			<td>PTAF10018B50</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>Hydrocortison</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>Brain endothelial media supplement</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>PeproTech</td>
			<td>200-01B</td>
			<td>Cytokine</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>11074547001</td>
			<td>Melanoma cell media supplement</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>Gibco</td>
			<td>11150-067</td>
			<td>Brain endothelial cell base media</td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>Deionized water</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td>Cytokine</td>
		</tr>
		<tr>
			<td>Transwell insert</td>
			<td>Corning</td>
			<td>CLS3464</td>
			<td>Porous membrane insert</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12604021</td>
			<td>Dissociation reagent</td>
		</tr>
	</tbody>
</table>

measuring changes in brain endothelial barrier integrity with two impedance-based biosensors in response to cancer cells and cytokines

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising pericytes, astrocytic foot processes, and tightly adhered endothelial cells. Here, the brain endothelial cells form the first line of barrier against blood-borne pathogens. In conditions like cancer and neuroinflammation, circulating factors in the blood can disrupt this barrier. Disease progression significantly worsens post barrier disruption, which permits access to or impairment of regions of the brain. This significantly worsens the prognoses, particularly due to limited treatment options available at the level of the brain. Hence, emerging studies aim to investigate potential therapeutics that can prevent these detrimental factors in the blood from interacting with the brain endothelial cells.
The commercially available Electric Cell-Substrate Impedance Sensing (ECIS) and cellZscope instruments measure the impedance across cellular monolayers, such as the BBB endothelium, to determine their barrier strength. Here we detail the use of both biosensors in assessing brain endothelial barrier integrity upon the addition of various stimuli. Crucially, we highlight the importance of their high-throughput capability for concurrent investigation of multiple variables and biological treatments.

Interpreting ECIS impedance data
Understanding optimal experimental conditions 
Herein the data can be directly viewed using the software (Figure 2A) or exported for analysis and graph plotting (Figure 2B). Figure 2A shows an example of data displayed on the actual software interface. The left graph shows an example of a disrupted connection due to improper lo...

Watch this Scientific Journal Video about Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines at JoVE.com

Author Spotlight: Studying Brain Endothelial Barrier in Metastatic Cancer Using Impedance-Based Biosensors

Here we demonstrate the technique of using impedance-based biosensors: ECIS and cellZscope, for measuring brain endothelial barrier strength. We detail the preparation and technique of adding various stimuli to an in vitro model of the brain endothelium. We measure, record, and give a representative analysis of the findings.

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising ...

measuring-changes-brain-endothelial-barrier-integrity-with-two

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416 Views.  University of Auckland. Here we demonstrate the technique of using impedance-based biosensors: ECIS and cellZscope, for measuring brain endothelial barrier strength. We detail the preparation and technique of adding various stimuli to an in vitro model of the brain endothelium. We measure, record, and give a representative analysis of the findings.

Video: Author Spotlight: Studying Brain Endothelial Barrier in Metastatic Cancer Using Impedance-Based Biosensors

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...