We thank National Cancer Institute IMAT program from NIH for funding this work [R33 CA174616-01A1].

1. Target Cell Preparation
<ol>
	<li>Maintain target cells (e.g. Raji lymphoma cells) in growth media (RPMI 1640 culture medium, with 10% heat-inactivated fetal bovine serum (FBS), 4nM L-glutamine, and 500 IU/ml penicillin/ streptomycin) at 37oC in a 5% CO2 incubator.</li>
	<li>Centrifuge the sample to pellet the target cells in a 15 ml tube. Centrifugation time varies with different cell types; centrifuge for 3 min at 468 x g for Raji cells.</li>
	<li>First aspirate any bubbles formed on surface of the supernatant, and remove the entire supernatant.</li>
	<li>Add 10 ml of PBS to the tube. Re-suspend the cells by pipetting the solution up and down several times to break up the cell clump.</li>
	<li>Centrifuge the sample for 3 min at 468 x g.</li>
	<li>Aspirate any bubbles formed on the surface of the supernatant, and remove the entire supernatant.</li>
</ol>
2. Preparation of Cell Microarrays
<ol>
	<li>Obtain cell attachment 96 well plate kits or single cell array 8 well chamber slides (see table of materials/reagents).</li>
	<li>Apply 1.2 &#956;l&#160;of the Activator to the DNA reagent from the kit. Cap the vial, invert it, and gently shake to mix the solution.</li>
	<li>Allow the solution to react for 20 min at RT.</li>
	<li>Apply the mixed solution to the target cells pellet (in the 15 ml tube).</li>
	<li>Add green fluorescence cytosolic dyes at a final concentration of 500 nM.</li>
	<li>Incubate the target cells with the mixed solution and the dye for 30 min at RT on an orbital shaker.</li>
	<li>Wash the cells twice with 5 ml PBS, and resuspend the cells in 10 ml growth media.</li>
	<li>Apply 100 &#181;l of the solution from step 2.7 to each well. Count the cell number by using a hemacytometer. Make sure cell numbers range between 5&#215;104 to 1&#215;106 cells per well.</li>
	<li>After 10-15 min of incubation at RT, gently wash the samples twice with 200-300 &#181;l growth media to remove any unbound cells. 
	NOTE: About 5-10 &#181;l growth media remains in each well after washing.</li>
</ol>
3. Whole Blood Cytotoxicity Assays
<ol>
	<li>Add 180 &#956;l of human whole blood to each sample well.</li>
	<li>Obtain Anti-CD20 antibody solution at 5 mg/ml, and perform serial dilution to make 50, 20, 10, 5, 2, 1, 0.5 &#956;g/ml of Anti-CD20 antibody solution in 1.5 ml tubes. Add 20 &#181;l of each concentration of Anti-CD20 antibody to each sample well making triplicates.</li>
	<li>Add Propidium iodide at a final concentration of 500 nM to each well.</li>
	<li>Incubate the resulting plate at 37&#176;C with 5% CO2 for 16 hr.</li>
	<li>Image the cells on the slides or 96 well plates with an automatic imaging system with 8X magnification.</li>
	<li>Use the cell counting program of the automatic imaging system to quantify the results. 
	NOTE: The software of the automatic imaging system provides simple step-by-step operations for cell counting. The protocol of using this software can be found at provider&#8217;s website.</li>
	<li>Select [Review Results] from the main page of the software.</li>
	<li>Select [The File Saved for The Samples].</li>
	<li>First gate FITC mean intensity &#62;50, then gate TxRed mean intensity &#62;20.</li>
	<li>Select [Plate Overview].</li>
	<li>Select [Export Tables]. 
	NOTE: The percentage cell killing formula of the program is shown below: 
	% target cell killing = (# of cells stained both green and red/ # of cells stained green)*100%</li>
</ol>

<ol>
	<li>Hutchinson, L., Kirk, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+drug+attrition+rates--where+are+we+going+wrong.">High drug attrition rates--where are we going wrong.</a> Nat Rev Clin Oncol. 8, 189-1890 (2011).</li><li>Moreno, L., Pearson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attrition+rates+be+reduced+in+cancer+drug+discovery.">Attrition rates be reduced in cancer drug discovery.</a> Informa healthcare. 8, 363-368 (2013).</li><li>Seok, 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=Inflammation+and+Host+Response+to+Injury,+Large+Scale+Collaborative+Research+Program.+Genomic+responses+in+mouse+models+poorly+mimic+human+inflammatory+diseases.">Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly mimic human inflammatory diseases.</a> Proc Natl Acad Sci U S A. 110, 3507-3512 (2013).</li><li>Suntharalingam, G., 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=Cytokine+storm+in+a+phase+1+trial+of+the+anti-CD28+monoclonal+antibody+TGN1412.">Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412.</a> N Engl J Med. 355, 1018-1028 (2006).</li><li>Eastwood, 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=Monoclonal+antibody+TGN1412+trial+failure+explained+by+species+differences+in+CD28+expression+on+CD4++effector+memory+T-cells.">Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells.</a> Br J Pharmacol. 161, 512-526 (2010).</li><li>Hsiao, S. C., Liu, H., Holstlaw, T. A., Liu, C., Francis, C. Y., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+assays+for+quantifying+cytotoxicity+with+single+cell+resolution.">Real time assays for quantifying cytotoxicity with single cell resolution.</a> PLoS One. 8, 10-1371 (2013).</li><li>Wang, S. Y., Weiner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rituximab:+a+review+of+its+use+in+non-Hodgkin&#8217;s+lymphoma+and+chronic+lymphocytic+leukemia.">Rituximab: a review of its use in non-Hodgkin&#8217;s lymphoma and chronic lymphocytic leukemia.</a> Expert Opin Biol Ther. 8, (2008).</li><li>Dalle, S., Thieblemont, C., Thomas, L., Dumontet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+in+clinical+oncology.">Monoclonal antibodies in clinical oncology.</a> Anticancer Agents Med Chem. 8, 523-532 (2008).</li><li>Brunner, K. T., Mauel, J., Cerottini, J. C., Chapuis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+of+the+lytic+action+of+immune+lymphoid+cells+on+51-Cr-labelled+allogeneic+target+cells+in+vitro;+inhibition+by+isoantibody+and+by+drugs.">Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs.</a> Immunology. 14, 181-196 (1968).</li><li>Korzeniewski, C., Callewaert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-release+assay+for+natural+cytotoxicity.">An enzyme-release assay for natural cytotoxicity.</a> J Immunol Methods. 64, (1983).</li><li>Gerlier, D., Thomasset, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+MTT+colorimetric+assay+to+measure+cell+activation.">Use of MTT colorimetric assay to measure cell activation.</a> Immunol Methods. 94, 57-63 (1986).</li><li>Toriello, N. M., 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=Integrated+microfluidic+bioprocessor+for+single-cell+gene+expression+analysis.">Integrated microfluidic bioprocessor for single-cell gene expression analysis.</a> Proc Natl Acad Sci U S A. 105, (2008).</li><li>Douglas, E. S., Hsiao, S. C., Onoe, H., Bertozzi, C. R., Francis, M. B., Mathies, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-barcode+directed+capture+and+electrochemical+metabolic+analysis+of+single+mammalian+cells+on+a+microelectrode+array.">DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.</a> Lab on a Chip. 9, 2010-2015 (2008).</li><li>Mayr, L. M., Bojanic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+trends+in+high-throughput+screening.">Novel trends in high-throughput screening.</a> Current opinion in pharmacology. 9, 580 (2009).</li><li>Zanella, F., Lorens, J. B., Link, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening:+seeing+is+believing.">High content screening: seeing is believing.</a> Trends in Biotechnology. 28, 237-245 (2010).</li><li>Coelho, J., P, L., Quinn, S., Murphy, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image+analysis+for+high-content+screening+and+analysis.">Automated image analysis for high-content screening and analysis.</a> J Biomol Screen. 15, 726-734 (2010).</li><li>Sundberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+and+ultra-high-throughput+screening:+solution-+and+cell-based+approaches.">High-throughput and ultra-high-throughput screening: solution- and cell-based approaches.</a> Current Opinion in Biotechnology. 11, 47 (2000).</li><li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine+is+more+than+genomic+medicine:+confusion+over+terminology+impedes+progress+towards+personalized+healthcare.">Personalized medicine is more than genomic medicine: confusion over terminology impedes progress towards personalized healthcare.</a> PERS MED. 9, 85 (2012).</li></ol>

Authors have no competing financial interests.

WCA is a critical in vitro anti-cancer screening tool with single cell resolution12-16, ideally utilized after traditional target screenings such as CDC and ADCC assays9-11, and before preclinical animal tests. Currently, primary target screening assays such as CDC or ADCC assays are all performed in a simplified media or a buffer system. However, drug candidates that show efficacy in these simplified buffer system are not always effective in the more complex whole blood system. Therefore, ...

Recent advances in the pharmaceutical industry have led to an increased interest in realizing the specific identifications of tumor cell antibodies and personalized cancer treatments; however, several obstacles are encountered in the process. Only 5% of agents that have anticancer activity in preclinical development are licensed after showing sufficient efficacy in phase II-III testing1,2. The preclinical strategies (both in vitro and in vivo) are suboptimal as many examples have shown that antitumor drugs behave differently in human and in laboratory animals mainly due to their different blood components3-5.
To address the necessity of an antitumor drug screening platform and to provide a check-point before costly animal experiments and clinical trials, a human whole blood cytotoxicity assay (WCA) is proposed for evaluating antitumor drug efficacy in a more relevant biological environment. The whole blood cytotoxicity assay can be used to evaluate the response of individual cells to antibodies and other drug candidates in human whole blood.
The WCA is developed by incorporating high-throughput cell positioning technology with high-throughput and high-content imaging6. By utilizing an automated imaging system, the number of both living and dead cells can be determined with a high degree of precision. Due to the fact that target cells are immobilized on the same focal plane, WCA is able to provide quantitative cytotoxicity analysis in real time without the removal of red blood cells. Moreover, the automatic imaging system provides several advantages such as that only target cells that have reached the specified criteria (e.g., fluorescently labeled cells, and cell morphology) are gated and processed. Also, it allows the production of 144 plates per day. Consequently, this imaging capability and throughput allows running of high-content and high-throughput experiments simultaneously. By combining WCA and the automated imaging system, high throughput quantitative cell cytotoxicity analysis can be achieved within a more biological relevant environment.

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:135px;">Name/Discription</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Cell attachment 96 well plate kits&#160;</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">AP9601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Suspension Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0801</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Adherent Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20+</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CCL-86</td>
			<td style="width:135px;">Raji cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20-</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL-8083</td>
			<td style="width:135px;">MC/CAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">RPMI 1640 with L-glutamine</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11875-119</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Thermo</td>
			<td style="width:119px;">SH30070.01HI</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Peni/Strep</td>
			<td style="width:172px;">Life Technologies</td>
			<td align="right" style="width:119px;">15070063</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cytosolic dye</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:135px;">Cell Tracker Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Rituxan (Biosimilar)&#160;</td>
			<td style="width:172px;">Eureka Therapeutics</td>
			<td style="width:119px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Human whole blood</td>
			<td style="width:172px;">Allcells</td>
			<td style="width:119px;">WB001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Propidium Iodide</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:119px;">P4170-10MG</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Automatic imaging system</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cell counting program</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
	</tbody>
</table>

real-time cytotoxicity assays in human whole blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

Anti-CD20 antibodies and lymphoma cells (Raji cells and MC/CAR) were chosen as a model system to demonstrate the whole blood cytotoxicity assay (WCA)7,8. Raji cells had high copy number of CD20 on the cell surface, while MC/CAR cells had low copy number of CD20 on their membrane. Targeted cells were first stained green with green fluorescence cytosolic dyes and arrayed on the 96-well plate. 10,000 - 50,000 target lymphoma cells were immobilized in each well. 180 &#181;l of freshly drawn human whole blood was a...

Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Real-time Cytotoxicity Assays in Human Whole Blood

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

real-time-cytotoxicity-assays-in-human-whole-blood

Research

JoVE Journal

Medicine

12.1K Views.  Adheren, Inc. The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis. 

Video: Real-time Cytotoxicity Assays in Human Whole Blood

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

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

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

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

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

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...