Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1. Preparation
<ol>
	<li>Place the real-time cell analyzer&#8217;s station in a tissue culture incubator set at 37 &#176;C and with 5% CO2. Carry out all cell culture handling and pharmacological treatments in a tissue culture hood under sterile conditions.</li>
	<li>NOTE: Neuronal cell lines requiring different temperature settings compared to standard conditions for culture, should be adjusted accordingly. For weakly adherent cell lines, use coating agents to facilitate the interaction of cells with the gold microelectrodes in the bottom of the wells of the E-Plate 96.</li>
	<li>Prepare 1,000X stock solutions of the pharmacological compounds for cell culture treatment (neuroprotective agents or second messenger pathways inhibitors in the appropriate solvent &#8211; dimethylsulfoxide or sterile water) and store them at -20 &#176;C. Use the solvent as vehicle in the following treatments.</li>
	<li>Culture human neuroblastoma SK-N-SH cells in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (proliferation medium) in cell culture flasks with 175 cm2 surface to density of about 75%. Keep cell passage number within a range (of about 10 passages) to ascertain consistency between experiments in terms of rate of cell proliferation and response to cytotoxicity. Lower passage numbers are preferred.</li>
	<li>Wash the adherent SK-N-SH cell layer with PBS, and trypsinize with 0.05% trypsin-EDTA solution for 5 min at 37 &#730;C. Centrifuge the cells at 170 &#215; g for 5 min to remove trypsin. Resuspend the cells in 10 ml proliferation medium. Count the cells with Scepter cell counter and adjust cell number to 300,000 cells/ml by diluting cells with proliferation medium.</li>
</ol>
2. Plating and Proliferation of SK-N-SH Cells
<ol>
	<li>Add 100 &#181;l proliferation medium to each well of the E-Plate 96 and leave it for 30 min in the tissue culture hood at RT to equilibrate. Insert the E-Plate 96 in the real-time cell analyzer station in the CO2 incubator at 37 &#176;C.</li>
	<li>Start the real-time cell analyzer software. On the Layout page select the wells included in the experiment and enter in the edit boxes the information about cell type, cell number, names and concentrations of chemical compounds used for cell treatment. 
	NOTE: For the purposes of this protocol at least 4 replicates are recommended for each treatment.</li>
	<li>On the Schedule software page determine &#8220;Steps&#8221; included in the experiment, by selecting the number of sweeps measuring Cell Index and the interval between sweeps for each step. Since Step 1 is predetermined for background measurement, select &#8220;Add a step&#8221; and set Step 2 to measure the Cell Index every 15 min for 96 hr. 
	NOTE: For treatments, in which effects with fast kinetics are expected, set sweeps at shorter intervals.</li>
	<li>Click Start to initiate the automatically predetermined Step 1 and measure the background impedance of the media. This value is automatically subtracted by the software at each data point once the cells are added to the wells.</li>
	<li>Use the previously prepared in step 1.5) cell suspension of 300,000 cells/ml in proliferation medium. 30,000 cells/well for cell toxicity/neuroprotection studies and 15,000 cells/well for cell proliferation studies. Take out the E-Plate and add 100 &#181;l cell suspension per well for cell toxicity/neuroprotection studies and add 50 &#181;l cell suspension and 50 &#181;l proliferation medium per well for cell proliferation studies. The number of cells plated per well needs to be optimized for each cell line empirically.</li>
	<li>Gently swirl the E-Plate 96 for even plating of the cells. Leave the E-Plate 96 for 30 min in the tissue culture hood at RT to allow the cells settle evenly to the bottom of the wells.</li>
	<li>Insert the E-Plate 96 in the real-time cell analyzer station and start Step 2 on the Schedule page. Monitor Cell Index values throughout the experiment by viewing the cell curves on the Plot page and the raw Cell Index data on the Cell Index page of the software.</li>
</ol>
3. Serum Deprivation
<ol>
	<li>24 hr after plating the cells, pause the experiment, and remove the E-Plate 96 from the real-time cell analyzer station. Dilute second messenger inhibitors from 1,000X stocks with DMEM/F12 serum-free medium &#8211; to 200&#215; the final concentration. Treat cells with 1 &#956;l inhibitors of second messenger pathways to be tested for their involvement in neurotoxicity/neuroprotection 30 min before initiating cytotoxicity, to inhibit the respective pathways. Return the E-Plate 96 to the station and resume experimental Cell Index measurements.</li>
	<li>After 30 min pause the experiment again. Carefully remove proliferation medium fully from the E-Plate 96 wells with a pipette (or multi-channel pipette), taking care not to disrupt the adherent cell layer by directing the edge of the pipette tip to the corner of the well. Add 200 &#181;l/well of serum-free DMEM/F12 medium (serum deprivation medium). Ensure rapid exchange of cell culture medium to minimize mechanical agitation of the cells.</li>
	<li>Dilute compounds to be tested for their neuroprotective effects from 1,000X stock with serum-free DMEM/F12 medium to 200&#215; the final concentration. Add 1 &#956;l compounds to be tested for their neuroprotective effects and 1 &#956;l inhibitors of second messenger pathways in the individual wells according to the experimental plan.</li>
	<li>In cells for proliferation studies do not change medium. Dilute compounds to be tested from 1,000X stock to 200&#215; the final concentration in proliferation medium, treat with 1 &#956;l per well and continue to culture in proliferation medium.</li>
	<li>Resume the experiment to continue with Cell Index measurements every 15 min for 96 hr as previously set.</li>
</ol>
4. Data Analysis
<ol>
	<li>For neurotoxicity/neuroprotection data normalize Cell Index to the last time point before pharmacological treatment or medium change to reduce variation between experiments by selecting &#8220;normalized Cell Index&#8221; and &#8220;normalize Time&#8221; on the Plot page.</li>
	<li>Highlight the wells on the Plot page for the experimental conditions of interest and click &#8220;Add&#8221; to plot the normalized Cell Index curves. Observe the kinetics of the neurotoxic/neuroprotective effect, or of second messengers inhibition as normalized Cell Index curves as a function of time.</li>
	<li>Observe the Cell Index curves for the tested drug treatments in proliferation medium as a function of time to assess whether an effect on proliferation is involved in the neuroprotective/neurotoxic effect.</li>
	<li>Export the experimental info for all Cell Index time points from the Cell Index software page into an Excel file to initiate statistical evaluation of the results 
	NOTE: As an initial step in the statistical analysis a set of modified MATLAB programs that use the Welch&#39;s t-test, with the p-value plotted semilogarithmically, utilize inverted axis against the time scale (provided as supplementary code files). These programs allow detection of p-values over the time-course of an entire real-time cell analyzer experiment, and thus aid the selection of time points for more detailed statistical analysis. See the supplementary material for detailed information on the programs.</li>
	<li>For statistical analysis of differences in Cell Index values under different treatment conditions at specific time points, select Cell Index values at respective time points from the exported Excel file. Analyze the Cell Index values for the selected time points with one-way or two-way analysis of variance (ANOVA) followed by a post-hoc test using statistical software.</li>
</ol>

<ol>
	<li>Cavallucci, V., D'Amelio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matter+of+life+and+death:+the+pharmacological+approaches+targeting+apoptosis+in+brain+diseases.">Matter of life and death: the pharmacological approaches targeting apoptosis in brain diseases.</a> Curr. Pharm. Des. 17 (3), 215-229 (2011).</li><li>Bull, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPA+CHaPD+001:+Review+of+Environmental+Chemicals+and+Neurotoxicity.">HPA CHaPD 001: Review of Environmental Chemicals and Neurotoxicity.</a> Focus on Neurological Diseases. , (2007).</li><li>Ke, N., Wang, X., Xu, X., Abassi, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+xCELLigence+system+for+real-time+and+label-free+monitoring+of+cell+viability.">The xCELLigence system for real-time and label-free monitoring of cell viability.</a> Methods Mol. Biol. 740, 33-43 (2011).</li><li>Limame, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+dynamic+cell+viability,+migration+and+invasion+assessments+by+novel+real-time+technology+and+classic+endpoint+assays.">Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.</a> PLoS One. 7 (10), e46536 (2012).</li><li>Diemert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+measurement+for+real-time+detection+of+neuronal+cell+death.">Impedance measurement for real-time detection of neuronal cell death.</a> J. Neurosci. Methods. 203 (1), 69-77 (2012).</li><li>Kustermann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+label-free,+impedance-based+real+time+assay+to+identify+drug-induced+toxicities+and+differentiate+cytostatic+from+cytotoxic+effects.">A label-free, impedance-based real time assay to identify drug-induced toxicities and differentiate cytostatic from cytotoxic effects.</a> Toxicol In Vitro. 27 (5), 1589-1595 (2013).</li><li>Marinova, Z., Walitza, S., Gr&#252;nblatt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT2A+serotonin+receptor+agonist+DOI+alleviates+cytotoxicity+in+neuroblastoma+cells:+role+of+the+ERK+pathway.">5-HT2A serotonin receptor agonist DOI alleviates cytotoxicity in neuroblastoma cells: role of the ERK pathway.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 44, 64-72 (2013).</li><li>Gonz&#225;lez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens+recruit+specific+cortical+5-HT(2A)+receptor-mediated+signaling+pathways+to+affect+behavior.">Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., Nakata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+(5-HT)+induces+glial+cell+line-derived+neurotrophic+factor+(GDNF)+mRNA+expression+via+the+transactivation+of+fibroblast+growth+factor+receptor+2+(FGFR2)+in+rat+C6+glioma+cells.">Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells.</a> J. Neurochem. 106 (1), 244-257 (2008).</li><li>Galante, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+toxicity,+conformation+and+morphology+of+typical+initial+aggregation+states+of+A&#946;1-42+and+A&#946;py3-42+beta-amyloids.">Differential toxicity, conformation and morphology of typical initial aggregation states of A&#946;1-42 and A&#946;py3-42 beta-amyloids.</a> Int. J. Biochem. Cell. Biol. 44 (11), 2085-2093 (2012).</li><li>Sheline, C. T., Cai, A. L., Zhu, J., Shi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+or+target+deprivation-induced+neuronal+death+causes+oxidative+neuronal+accumulation+of+Zn2+and+loss+of+NAD.">Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+and loss of NAD.</a> Eur. J. Neurosci. 32, 894-904 (2010).</li></ol>

Publication costs for the current video-article were sponsored by &#8220;ACEA Biosciences&#8221;.

The current protocol presents the utility of a real-time cell analyzer to assess continuously and under label-free conditions the neuroprotective/neurotoxic effects of compounds in neuronal cell lines and to gain insight into the second messenger pathways involved in the effect.
Even though the real-time cell analyzer&#8217;s utility to study cytotoxicity and effects of drugs on cell proliferation is generally recognized, only a few studies have used it in neuronal related cell types. We have previously shown the utility of the real-time cell analyzer to monitor neuroprotection of a 5-HT2A receptor agonist in human neuroblastoma SK-N-SH cells, while Galante et al., have shown it can be used to assess neurotoxicity of beta-amyloid peptides in human neuroblastoma SH-SY5Y cells7,10. A recent study has suggested that while the real-time cell analyzer measured reliably cell proliferation and cell death in a neuronal cell line and neuronal precursor cells, its precision in assessing cell death in primary neurons depended on the severity of the insult5. Importantly, the alternating current used for the real-time cell analyzer system is of very low magnitude, generating microampere currents. These currents are well below the threshold potential of excitable cells as neurons. The real-time cell analyzer system relies on impedance measurements from the bottom surface of the E-Plate 96 wells, therefore the degree of cell adherence is of critical importance. It is possible that further optimization of coating of the E-Plate wells with extracellular matrix proteins for better adherence may improve results obtained with primary neurons.
The current manuscript demonstrates a novel approach of using the real-time cell analyzer system to delineate second messenger pathways involved in neuroprotection. Our protocol relies on the chemical inhibition of second messengers hypothesized to be involved in the action of a neuroprotective agent. Table 1 lists some commonly used second messenger pathways inhibitors, which can be relevant to the 5-HT2A receptor as an example, and their concentrations suitable for the presented experimental paradigm. This experimental design allows for fast screening of potentially relevant second messenger pathways, to select downstream targets for more detailed studies through complementary approaches.
An important advantage of the real-time cell analyzer is also the ability to assess whether an effect on proliferation is a part of an observed neuroprotective effect. Using neuronal cell lines instead of primary neurons in toxicity assays offers some advantages, as lower cost, easy access and no need for animal experimentation. However, due to the fact that neuronal cell lines in contrast to primary neurons proliferate, potential effect on proliferation needs to be controlled for in neurotoxicity assays. The opportunity the real-time cell analyzer offers to perform this control in the same experimental E-plate 96, facilitates the experimental design.
Several technical parameters need to be considered in experiments with the real-time cell analyzer. The number of cells to be plated per well should be determined empirically through titration experiment for each tested cell type. Cytotoxicity and proliferation treatment are all started after 24 h of initiation of cell culture, to allow full adherence of the cells. Optimal results in cytotoxicity experiments starting when cells have reached 65-70% confluence were obtained in the current protocol. For proliferation experiments lower cell confluence is preferred (20-30%) to follow the effect of a drug on a proliferation curve.
In the current protocol serum deprivation neurotoxicity paradigm is presented due to its robustness, ease of use and utility to approximate trophic deprivation mediated neuronal death, which is important during development, acute brain or nerve trauma, and neurodegeneration11. However, a wide range of neurotoxicity paradigms can be used with the real-time cell analyzer system. Here, co-treatment with a cytoprotective agent at the time of cytotoxicity is presented. However, pretreatment or treatment after the toxic event (assessing cell recovery) can also be employed depending on the compounds being tested.
Overall, the current protocol suggests the utility of a real-time cell analyzer system to assess neurotoxicity and neuroprotection in neuronal cell lines continuously and label-free and to gain insight into downstream pathways involved in these effects.

Neuronal cell death plays a critical role in the pathophysiology of many brain-related disorders1. The availability of reliable and high-throughput in vitro toxicity assays is critical to gain better insight into the mechanisms of neurotoxicity and to help select neuroprotective molecules as therapeutic candidates in drug development2. However, there are many limitations to most widely used in vitro neurotoxicity assays.They assess neurotoxicity/neuroprotection at a single time-point not allowing kinetic resolution; often use label or probe which can interfere with the signaling pathways and limit additional studies in the same cell population, and are often labor-intensive, and in many cases do not provide mechanistic insight. In the present study we demonstrate the utility of a real-time impedance-based cell analyzer to determine neurotoxicity and neuroprotection in a neuronal cell line in real-time and under label-free conditions and to provide insight into downstream mechanisms through analysis of second messenger pathways involved in the effect.
Previous studies have confirmed the validity of the real-time cell analyzer to determine cytotoxicity as well as effects on cell proliferation in cell lines in comparison with standard techniques3,4,5,6. For example, a good correlation was observed between readouts of the standard cell viability WST-1 assay and Cell Index values at several time points under basal proliferation conditions and after two different toxic paradigms in HeLa cells3. In A549 and MDA-MB-231 cells proliferation and cytotoxicity provoked with the microtubule stabilizer paclitaxel showed very similar values when assessed by Cell Index measurements and the standardly used sulforhodamine B (SRB) assay4. In the neuronal cell line of immortalized hippocampal neurons HT-22 Cell Index measurements were validated for their ability to detect cell proliferation, glutamate cytotoxicity and cytoprotection against the widely used 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium-bromide (MTT) assay5. In the same study the MTT assay results and Cell Index measurements also correlated well in measuring neuronal progenitor cells proliferation, cytotoxicity after growth factors deprivation and rescue of cytotoxicity by the pan-caspase inhibitor QVD5. Cytotoxicity induced in NIH 3T3 cells by Vandetanib (vascular endothelial growth factor receptor and epidermal growth factor receptor inhibitor) showed similar results measured with Cell Index values or neutral red uptake assay6.
We have recently used the real-time cell analyzer system to assess neuroprotective effects of the serotonin 2A (5-HT2A) receptor agonist (&#177;)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) in a neuronal cell line (SK-N-SH cells) and screened for the involvement of second messenger pathways through monitoring the effect of their chemical inhibition on the observed neuroprotection7. Interestingly, the 5-HT2A receptor has both hallucinogenic and nonhallucinogenic agonists (like DOI and lisuride, respectively), which may activate both common and distinct second messenger pathways8.
The advantages of the presented technique are that it allows to collect real-time information on cell survival in the course of days, to delineate second-messenger pathways involved, to assess the possible contribution of proliferation effects to neuroprotection, and to select an optimal time for additional end-point studies on the same cell population. A schematic diagram of the workflow in the current protocol is presented in Figure 1.

<table><tbody><tr><td>xCELLigence&amp;nbsp; RTCA SP system bundle</td><td>ACEA Biosciences</td><td>No: 00380601030</td><td>Consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td></tr><tr><td>E-plate 96</td><td>ACEA Biosciences</td><td>No: 05232368001</td><td>For culturing the cells, inserted in the RTCA SP Station</td></tr><tr><td>SK-N-SH cells</td><td>ATCC (in partnership with LGC Standards)</td><td>HTB-11</td><td>Can be replaced by another adherent neuronal cell line of interest</td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8437</td><td>Cell culture medium</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>16140-063</td><td>Supplements proliferation, but not serum deprivation medium</td></tr><tr><td>Tissue culture flask T175</td><td>Sarstedt</td><td>83.1812.302</td><td>For culturing cells, which will be later plated on the E-Plate 96</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td>For trypsinization of cells cultured in tissue culture flasks</td></tr><tr><td>Scepter 2.0 cell counter</td><td>Merck Millipore</td><td>PHCC20060</td><td>Automated cell counter</td></tr><tr><td>Phosphate-buffered saline</td><td>Life Technologies</td><td>10010-015</td><td>For washing the cells</td></tr><tr><td>(&amp;plusmn;)-DOI hydrochloride</td><td>Sigma-Aldrich</td><td>D101</td><td>5-HT2A agonist for cell culture treatment</td></tr><tr><td>LY-294002 hydrochloride</td><td>Sigma-Aldrich</td><td>L9908</td><td>PI3-K inhibitor for cell culture treatment&amp;nbsp;</td></tr><tr><td>Lisuride maleate</td><td>Tocris Bioscience</td><td>4052</td><td>Compound with 5-HT2A agonistic activity for cell culture treatment</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D4540</td><td>For dissolving LY-294002 and lisuride maleate</td></tr></tbody></table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Serum deprivation leads to decrease in Cell Index values, which can be monitored continuously with the real-time cell analyzer
Neurotoxic stimuli to the cells lead to a decrease in Cell Index values, which can be monitored in real-time with the presented technique and the dynamics of which is dependent on the specific neurotoxic stimulus and the cell type studied. Figure 2 demonstrates the increase in Cell Index when SK-N-SH cells are grown in proliferation medium until reaching a plateau (green line) and the drop of Cell Index followed by a stabilization at the new lower levels after switching the cells to serum deprivation medium (red line). This drop may represent a change in cellular adhesion due to withdrawal of the serum.
Neuroprotective effects of drug treatment can be monitored continuously with the real-time cell analyzer
The neuroprotective effect of compounds added before (pre-treatment), at the same time (co-treatment) or after neurotoxic stimulus (suggesting cell recovery) can also be followed continuously with the real-time cell analyzer, thus providing information about the rate and duration of the neuroprotective effect. Figure 3 shows representative results of the dynamics of neuroprotection of two 5-HT2A agonists - 5 &#181;M DOI (Figure 3A)&#160;and 20 &#181;M lisuride (Figure 3B)&#160;added at the time of switching cells into serum deprivation medium.
The real-time cell analyzer allows to differentiate between effects on cell survival and cell proliferation
An important question in neuroprotection studies in neuronal cell lines is whether an effect on cell proliferation contributes to the observed compound effect on cell survival. With the real-time cell analyzer the effect on proliferation can be tested in the same E-Plate 96 assessing neurotoxicity. Figure 4 shows the lack of effect of 5 &#181;M DOI treatment on SK-N-SH cells proliferation, suggesting an effect on proliferation does not contribute to the observed neuroprotective effect.
The real-time cell analyzer allows to determine which second messenger pathways are involved in neuroprotective/neurotoxic effects
Receptor activation initiates a cascade of second messengers effects. The current protocol allows to screen for the involvement of a number of second messengers in a neuroprotective/neurotoxic effect in real-time through pretreatment with their inhibitors. Since the effect of second messengers inhibitors on cell survival can be pronounced, and is in many cases not linear with time, following its kinetics is very informative. Figure 5 presents the effect of 10 &#181;M phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 on cell survival and DOI neuroprotection in serum-deprived SK-N-SH cells. In the presented graph, 10 &#181;M LY294002 prevented the neuroprotective effect of DOI in serum deprivation, but also had some toxic effect of its own.
Relevant second messenger pathways and inhibitor concentrations for each tested compound can be determined through literature search. Concentrations suitability of second messengers inhibitors has to be verified, and if needed optimized experimentally. The goal of optimization is to select a concentration, which has a minimal effect on Cell Index values by itself, while still inhibiting effectively the respective second messenger. As an example, some second messenger pathways inhibitors, which can be relevant to the 5-HT2A receptor, are presented in Table 1. These inhibitors have been previously used to assess downstream targets involved in serotonergic increase of the extracellular signal-regulated kinase phosphorylation9. We have shown their utility to assess downstream pathways involved in neuroprotection by 5-HT2A receptor agonists after confirming and in some cases optimizing their concentrations for our experimental paradigm7.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51748/51748fig1highres.jpg" width="500" /> 
Figure 1. Schematic diagram of the workflow presented in the current protocol. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51748/51748fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51748/51748fig2highres.jpg" width="500" /> 
Figure 2. Effect of serum deprivation on Cell Index. Serum deprivation (red line) induces a rapid decrease in Cell Index, compared to the gradual increase in Cell Index values in proliferation medium (green line). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51748/51748fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51748/51748fig3highres.jpg" width="500" /> 
Figure 3. Effect of 5-HT2A agonists on Cell Index after serum deprivation. Treatment with cytoprotective drugs (in this experiment 5-HT2A agonists DOI (5 &#181;M) (A) (magenta line) and lisuride (20 &#181;M) (B) (dark magenta line) partially restores Cell Index values decreased by serum deprivation (red line). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51748/51748fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51748/51748fig4highres.jpg" width="500" /> 
Figure 4. Cell Index values in the course of cell proliferation. Treatment with drugs in proliferation medium (in the current experiment 5 &#181;M DOI) (magenta line) allows to assess the effect on proliferation compared to vehicle treatment (green line). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51748/51748fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51748/51748fig5highres.jpg" width="500" /> 
Figure 5. Second messenger inhibitors may affect cell survival on their own and can be used to screen for the effect of second messenger pathways on observed neuroprotection. In this experiment cells were subjected to serum deprivation being treated with vehicle (red line), 1 &#181;M DOI (magenta line), pretreated with 10 &#181;M PI3-K inhibitor LY294002 and treated with 1 &#181;M DOI (blue line) or pretreated with 10 &#181;M LY294002 and treated with vehicle (black line). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51748/51748fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" fo:content-width="4in" src="/files/ftp_upload/51748/51748table1.jpg" width="400" /> 
Table 1: Examples of second messenger inhibitors, which can be relevant for 5-HT2A receptor downstream pathways and their concentrations used by us in real-time cell analyzer experiments.

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

real-time-impedance-based-cell-analyzer-as-tool-to-delineate

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.4K Views.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Video: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Environment

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

Biochemistry

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Developmental Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biology

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Cancer Research

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells' Response to Metal-Organic Frameworks 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

Bioengineering

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Impedance-Based Biosensors in Investigating Brain Endothelial Barrier Strength

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.

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.

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