We thank Drs. John Hancock, Ray Grill, Andrew Morris and Olga Chumakova for their support. The FlexStation 3 system is part of the UTHSC Cytodynamic Imaging Facility. We also thank Drs. Ardem Patapoutian and Michael Bandell from the Scripps Research Institute and Genomics Institute of the Novartis Research Foundation (GNF) for providing the HEK293-hTRPA1 stable cell line. The research is supported in part by grants from NIH (GM081658 to MXZ) and Texas Medical Center Digestive Diseases Center (to HH) and Mission Connect/TIRR Foundation (to HH).

1. Cell preparations in 96-well plates
For HEK293 cells transiently trasfected with human TRPA1 cDNA
<ol>
<li>HEK293 cells are grown for one to two days in Dulbecco's Minimal Essential Medium (DMEM) containing 4.5 mg/ml glucose (Thermo Scientific, SH3002201), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, 16000-044), 50 units/ml penicillin and 50 &mu;g/ml streptomycin (Invitrogen, 15140-163). On the day of the transfection, the cell density should reach 70-90%.</li>
<li>Coat 96-well plates by pipetting 50 &mu;l poly-L-ornithine (Sigma P3655, 20 &mu;g/ml in sterile distilled water) to each well and incubating at 37&deg;C for at least 15 min. Rinse each well once with Dulbecco's Phosphate Buffered Saline (DPBS) without Mg2+ and Ca2+ (Thermo Scientific, SH3002803). The plate may be left in the cell culture hood for a few hours.</li>
<li>For every well of a 96-well plate, dilute in a 1.5-ml Eppendorf tube 0.2 &mu;g cDNA in 25 &mu;l OPTI-MEM (Invitrogen, 31985-070). Scale up proportionally according to the number of wells to be transfected by the same cDNA. Also dilute in a separate 1.5-ml Eppendorf tube 0.4 &mu;l Lipofectamine 2000 (Invitrogen, 11668-019) in 25 &mu;l OPTI-MEM for each well to be transfected. Scale this up according to the total number of wells to be transfected.</li>
<li>Briefly vortex the diluted cDNA and Lipofectamine 2000 and let them sit at the room temperature for about 5 min.</li>
<li>Transfer an equal volume of the diluted Lipofectamine 2000 to the tube that contains the diluted cDNA, vortex and let the mixture sit at room temperature for 15 min to 2 hrs. During this time, transfer the cDNA/Lipofectamine 2000 mixture at 49 &mu;l/well to the poly-L-ornithine coated plate.</li>
<li>Trypsinize the HEK293 cells (Step 1.1), count cell density using a hemocytometer or Countess automated cell counter (Invitrogen, C10227), and transfer a desired amount of cells (generally about ~125,000-150,000 cells/well) to a 15-ml sterile conical-bottom centrifuge tube. Centrifuge at 1000 x RPM for 5 min. Resuspend cells at ~1,250,00-1,500,000 cells/ml in the DMEM culture medium, supplemented with 10% heat-inactivated fetal bovine serum, but without antibiotics.</li>
<li>Dispense 98 &mu;l cell suspension to each well that contains the cDNA-Lipofectamine 2000 mixture using a multichannel pipetter or the MicroFill Microplate Dispenser (Bio-Tek). Let the plate sit in the hood for at least 30 min before placing it to the humidified cell culture incubator. Incubate at 37&deg;C, 5% CO2 for 20-44 hrs.</li>
</ol>
For HEK293 cells stably expressing TRPA1
<ol start="8">
<li>Follow Step 1.2 to coat 96-well plates.</li>
<li>The inducible HEK293-hTRPA1 cell line was generously provided by Dr. A. Patapoutian (The Scripps Research Institute) and maintained in the same medium as the wild-type HEK293 cells (Step 1.1) but supplemented with 5 &mu;g/ml blasticidin (Invitrogen, A1113902) and 50 &mu;g/ml hygromycin B (Invitrogen, 10687010). When cell density reaches about 90%, trypsinize cells, count cell density, and centrifuge the desired amount of cells at 1000 x RPM for 5 min. Resuspend cells at a density of ~1,000,000 cells/ml in the same culture medium supplemented with doxycycline (Fisher, BP26535, 0.25 &mu;g/ml).</li>
<li>Dispense the cell suspension to poly-L-ornithine coated plates at 100 &mu;l/well using a multichannel pipetter or the MicroFill (Bio-Tek). Let the plate sit in the hood for at least 30 min before placing it to the humidified cell culture incubator. Incubate at 37 &deg;C, 5% CO2 for 16 - 24 hr.</li>
</ol>
2. Solutions
<ol>
<li>Hank's buffered salt solution (HBSS) is used as the extracellular solution. It contains (in mM): 137 NaCl, 5.4 KCl, 0.25 Na2HPO4, 0.44 KH2PO4, 1.3 CaCl2, 1.0 MgSO4, 1.0 MgCl2, 10 glucose, 10 HEPES, with pH adjusted to 7.4 using 1N NaOH).</li>
<li>Fluo-4 assay buffer contains 2 mM probenecid in HBSS. Make 250 mM stock probenecid (Sigma, P8761) in 0.5 N NaOH. Dilute to HBSS at a ratio of 1:125. Readjust pH to 7.4 using 1 N HCl.</li>
</ol>
3. Cell loading with Fluo-4 AM
<ol>
<li>Dissolve 1 mg Fluo-4 AM (Invitrogen, F14202) in 912 &mu;l anhydrous DMSO to make a stock solution of 1 mM. The Fluo-4 AM stock solution can be stored at -20 &deg;C for at least one month. </li>
<li>Mix 10 &mu;l 1 mM Fluo-4 AM with 10 &mu;l Pluronic F-127 (Invitrogen, P3000MP, 20% (w/v) solution in DMSO) and then transfer the entire content to 5 ml Fluo-4 assay buffer (Step 2.2) supplemented with 0.1% Bovine serum albumin (Sigma, A9418).</li>
<li>Wash cells grown in 96-well plates (Step 1.7 or 1.10) with HBSS at 80 &mu;l/well 2 times using a microplate washer (such as ELx405TM from Bio-Tek).</li>
<li>Add the Fluo-4 AM loading solution (Step 3.2) at 50 &mu;l/well using a multichannel pipetter or the MicroFill (Bio-Tek) and incubate the plate at 37 &deg;C for 1 hr.</li>
<li>Wash cells 3 times with the Fluo-4 assay buffer (Step 2.2.) using the ELx405TM microplate washer. After the last wash, leave 80 &mu;l of the Fluo-4 assay buffer in each well.</li>
</ol>
4. Preparation of compound plate
<ol>
<li>During the 1 hr cell loading period, prepare a series of 3x dilutions of agonists (or antagonists) of 3x of the desired final concentrations in the Fluo-4 assay buffer.</li>
<li>Pipette 250-300 &mu;l of the diluted compound solutions to a 96-well compound plate. Arrange to have the serial dilutions of a compound and the corresponding positive and negative controls in the same column of the compound plate whenever it is possible because FlexStation 3 reads one single column at a time.</li>
</ol>
5. Plate reading in FlexStation 3
<ol>
<li>Turn on the system (FlexStation 3, computer, and the SoftMax Pro 5.2 software)</li>
<li>Save the experiment with a new file name. At the set-up, choose FLEX mode and enter the following values:</li>
</ol>
Protocol #1, agonist effect 
<img src="/files/ftp_upload/3149/3149table1.jpg" alt="Table 1" />
Protocol #2, antagonist effect 
<img src="/files/ftp_upload/3149/3149table2.jpg" alt="Table 2" />
<ol start="3">
<li>Place a new tip box, compound plate and the reading plate to the appropriate trays in FlexStation 3 and click "read" button. The FlexStation 3 will read one column at a time and add compounds at the preprogrammed time points without interrupting the reading. It takes about 3 min (5 min for Protocol #2) to finish reading one column and the machine will continue to read the next column with compound additions performed as programmed by the user in step 5.2 until all columns are read. </li>
</ol>
6. Data analysis
<ol>
<li>Experimental data may be processed directly using the SoftMax Pro 5.2 software or copied and pasted into any spreadsheet program, such as Microsoft Excel. Concentration-response curve of a TRPA1 agonist flufenamic acid (FFA) is constructed using the following least-squares fitting routine. <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="Equation 1" />, where Y is the observed response, Ymax is the normalized maximal response, C is the corresponding drug concentration, EC50 is the concentration that evoked a half-maximal response, and nH is the apparent Hill coefficient. Concentration-inhibition curve for a putative TRPA1 antagonist compound A is obtained by using a fixed concentration of FFA (100 &mu;M), and progressively increasing the concentration of compound A. IC50 value of compound A is calculated by least-squares fitting to the following equation. <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="Equation 2" />, where Y is the observed response and Ymax is normalized peak response, [Ant] is the corresponding compound A's concentration, IC50 is the antagonist concentration that produces half-maximal inhibition of FFA-evoked response, and nH is the apparent Hill coefficient.</li>
</ol>
7. Representative Results:
TRPA1 is a Ca2+-permeable cation channel, activation of which leads to calcium influx that can be detected by the Ca2+-sensitive dye, Fluo-4 (1-2). A HEK293-hTRPA1 stable cell line was used to study the concentration-response relationships of TRPA1 agonists and antagonists. Cells were loaded with Fluo-4 AM, placed in 80 &mu;l of the assay buffer, and read using FlexStation 3. After recording the baseline fluorescence for 30 s, 40 &mu;l of a serial dilution of the TRPA1 agonist, FFA, each at 3x of the desired final concentration, were added to the cells through a multi-channel pipetter included as a part of the fluidics module of FlexStation 3. The fluorescence changes were monitored for an additional 180 s. The relative Fluo-4 fluorescence changes for each well were expressed as &Delta;F= (F - F0), where F and F0 are fluorescence values at the time of interest and the beginning of the reading, respectively. Representative traces of the time courses to fluorescence changes in response to a series of FFA concentrations are shown in Fig. 1. Note for the last four FFA concentrations, although the maximal response levels are similar, the activation kinetics is clearly faster at the higher than at the lower FFA concentrations. Therefore, to distinguish these differences, the concentration - response relationship was constructed by measuring the initial rates (slope) of the fluorescence increase upon addition of varying concentrations of FFA (Fig. 2). The half maximal effective concentration (EC50) of FFA was determined to be 55.4 &mu;M (n=3). To test the putative TRPA1 antagonist, compound A, we performed a similar experiment using protocol #2 (Step 2). In this case, a series of dilutions of the antagonist were added at 30 s (40 &mu;l of 3x desired final concentrations) and the FFA was added 180 s later (60 &mu;l at 300 &mu;M for a final concentration of 100 &mu;M). Compound A dose-dependently reduced the response of the TRPA1 cells to FFA with a half maximal inhibitory concentration (IC50) of 4.3 &mu;M (n=3) (Fig. 2).
<img src="/files/ftp_upload/3149/3149fig1.jpg" alt="Figure 1"/> Figure 1. Concentration-dependent activation of human TRPA1 by FFA. Traces are FFA-evoked increase of fluorescence over time (only responses in the first 60 s are shown to better illustrate the fast kinetics of the FFA-evoked responses). Please note that at higher concentrations FFA evoked fluorescence increase with a much faster kinetic than at lower concentrations, even though the peak fluorescence levels were similar. 
<img src="/files/ftp_upload/3149/3149fig2.jpg" alt="Figure 2"/> Figure 2. Concentration - response relationships for FFA-induced fluorescence change and compound A-induced inhibition of FFA response in human TRPA1 stably expressed in HEK293 cells. &Delta;,The concentration - response relationship of FFA was determined by measuring the initial rates (slope) of the normalized fluorescence increase upon addition of varying concentrations of FFA. O, Concentration-response relationship of inhibition of FFA-evoked calcium response by a putative TRPA1 antagonist, compound A.

<ol>
	<li>Hu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPA1+channels+by+fenamate+nonsteroidal+anti-inflammatory+drugs.">Activation of TRPA1 channels by fenamate nonsteroidal anti-inflammatory drugs.</a> Pflugers Arch. 459, 579-592 (2010).</li><li>Hu, H., Bandell, M., Petrus, M. J., Zhu, M. X., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+activates+damage-sensing+TRPA1+ion+channels.">Zinc activates damage-sensing TRPA1 ion channels.</a> Nat Chem Biol. 5, 183-190 (2009).</li><li>Perney, T. M., Hirning, L. D., Leeman, S. E., Miller, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+calcium+channels+mediate+neurotransmitter+release+from+peripheral+neurons.">Multiple calcium channels mediate neurotransmitter release from peripheral neurons.</a> Proc Natl Acad Sci. 83, 6656-6659 (1986).</li><li>Mason, S. A., MacLeod, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+action+potential+duration+and+calcium+regulation+in+males+and+females.">Cardiac action potential duration and calcium regulation in males and females.</a> Biochem Biophys Res Commun. 388, 565-570 (2009).</li><li>Raat, N. J., Wetzels, G. E., DeMey, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-contraction+relationship+in+rat+mesenteric+arterial+smooth+muscle.+Effects+of+exogenous+and+neurogenic+noradrenaline.">Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline.</a> Pflugers Arch. 436, 262-269 (1998).</li><li>Alagarsamy, S., Johnson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+calcium+channel+involvement+in+NMDA-induced+activation+of+NOS.">Voltage-dependent calcium channel involvement in NMDA-induced activation of NOS.</a> Neuroreport. 6, 2250-2254 (1995).</li><li>Su, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM7+regulates+cell+adhesion+by+controlling+the+calcium-dependent+protease+calpain.">TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain.</a> J Biol Chem. 281, 11260-11270 (2006).</li><li>Dubinsky, J. M., Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+concentrations+during+"chemical+hypoxia"+and+excitotoxic+neuronal+injury.">Intracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury.</a> J Neurosci. 11, 2545-2551 (1991).</li><li>George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+channel+activation+augments+NMDA+receptor+function+and+promotes+neurite+outgrowth+in+immature+cerebrocortical+neurons.">Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons.</a> J Neurosci. 29, 3288-3301 (2009).</li><li>Price, K. L., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FlexStation+examination+of+5-HT3+receptor+function+using+Ca2++-+and+membrane+potential-sensitive+dyes:+advantages+and+potential+problems.">FlexStation examination of 5-HT3 receptor function using Ca2+ - and membrane potential-sensitive dyes: advantages and potential problems.</a> J Neurosci Methods. 149, 172-177 (2005).</li><li>Laude, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+assays+of+recombinant+IP3+receptors.">Rapid functional assays of recombinant IP3 receptors.</a> Cell Calcium. 38, 45-51 (2005).</li><li>Marincsak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analgesic+drug,+tramadol,+acts+as+an+agonist+of+the+transient+receptor+potential+vanilloid-1.">The analgesic drug, tramadol, acts as an agonist of the transient receptor potential vanilloid-1.</a> Anesth Analg. 106, 1890-1896 (2008).</li><li>Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+high+throughput+screening+assays+against+three+subtypes+of+Ca(v)3+T-type+channels+using+molecular+and+pharmacologic+approaches.+Assay+Drug+Dev+Technol.">Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches. Assay Drug Dev Technol.</a> 5, 191-203 (2007).</li><li>Christensen, B. N., Kochukov, M., McNearney, T. A., Taglialatela, G., Westlund, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-sensing+G+protein-coupled+receptor+mobilizes+calcium+in+human+synovial+cells.">Proton-sensing G protein-coupled receptor mobilizes calcium in human synovial cells.</a> Am J Physiol Cell Physiol. 289, C601-C608 (2005).</li><li>Boulay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+a+novel+mammalian+homolog+of+Drosophila+transient+receptor+potential+(Trp)+involved+in+calcium+entry+secondary+to+activation+of+receptors+coupled+by+the+Gq+class+of+G+protein.">Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein.</a> J Biol Chem. 272, 29672-29680 (1997).</li><li>Nanda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+screening+of+adrenergic+receptors+by+measuring+intracellular+calcium+using+the+FlexStation+scanning+fluorimeter.">Functional screening of adrenergic receptors by measuring intracellular calcium using the FlexStation scanning fluorimeter.</a> Biotechnol J. 4, 417-422 (2009).</li></ol>

No conflicts of interest declared.

Intracellular Ca2+ plays important roles in many physiological and pathological conditions, such as neurotransmitter release (3), cardiac action potential (4), muscle cell contraction (5), cell signal transduction (6-7), and excitotoxic cell death (8). Measurement of intracellular Ca2+ levels helps to elucidate functional changes of Ca2+-permeable channels in the plasma membrane or intracellular organelles and the contribution of Ca2+ to the above processes (9-13). The as...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>FlexStation 3		</td>
			<td>Molecular Devices</td>
			<td>FLEX3</td>
			<td>&nbsp;</td>
		</tr>		
		<tr>
			<td>96-Well, FlexStation Pipet Tips</td>
			<td>Molecular Devices</td>
			<td>9000-0912</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-Well Plates</td>
			<td>Greiner Bio-one</td>
			<td>655090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-Well Clear Flat Bottom plates		</td>
			<td>Costar</td>
			<td>3595</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM		</td>
			<td>Invitrogen</td>
			<td>12430-104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Opti-MEM		</td>
			<td>Invitrogen</td>
			<td>31985-070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000	</td>
			<td>Invitrogen</td>
			<td>	11668-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluo-4 AM	</td>
			<td>Invitrogen</td>
			<td>	F14202</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PluronicF-127		</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma</td>
			<td>	P36551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>probenecid	</td>
			<td>Sigma</td>
			<td>	P8761</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin	</td>
			<td>Sigma</td>
			<td>	A9418</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

cell-based-calcium-assay-for-medium-to-high-throughput-screening-trp

Research

JoVE Journal

Bioengineering

21.0K Views.  The University of Texas Health Science Center at Houston. This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Video: Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...