Mrs. Alster, Mr. Honrath and Dr. Jiang are appreciated for their outstanding technical assistance. Also appreciated is Dr. Volz and the research group of Dr. Dandri for providing the access to their digital PCR device.

1. Preparing Mix Culture Containing Both Tumor Cells and Non-tumor Cells
<ol>
	<li>Use tumor cells from an established cell line MPNST S4625. Use fibroblasts as the non-tumor cells6,7</li>
	<li>Culture both tumor cells and fibroblasts in standard DMEM medium with 10% fetal bovine serum (BSA), 3 mM glutamine, 0.1 mM 2-mercaptoethanol, 500 U/ml penicillin, 500 g/ml streptomycin and 1 mM sodium pyruvate at 37 &#176;C and 5% CO25-7. At sub-confluence, harvest the cells with 0.05% trypsin and count them using a hemocytometer.</li>
	<li>Mix 400 tumor cells and 1,600 non-tumor cells together in each well of a 96-well culture plate in 0.2 ml medium. Considering faster growth of tumor cells over the treatment period of 5 days, the initial proportion of tumor cells was set at 25%. Set up 4 replicates for each drug concentration. Incubate the cells at 37 &#176;C and 5% CO2 O/N.</li>
</ol>
2. Drug Treatment
<ol>
	<li>Use nilotinib as the test drug. Prepare stock solutions of 0, 2, 4 and 20 mM nilotinib in DMSO. Dilute each of them by 200-fold in 5 ml DMEM medium, giving 2-fold concentrated drug-solutions of 0, 10, 20 and 100 &#181;M6.7.</li>
	<li>Remove 0.1 ml medium from each well and add 0.1 ml of medium containing drug. The final concentrations are 0, 5, 10 and 50 &#181;M. Continue incubating the cells in medium containing drug for 5 days.</li>
	<li>At the end of the treatment, inspect the attached cells using a phase-contrast microscope and take photos. Aspirate the medium away, wash the survived adhesive cells once with 0.2 ml PBS.</li>
</ol>
3. Preparing DNA for Digital PCR
<ol>
	<li>Lyse cells using HotShot8.
	<ol>
		<li>Add 50 &#181;l alkaline lysis solution (25 mM NaOH, 0.2 mM EDTA) to each well. Seal the wells with foil. Incubate the plate at 95 &#176;C in an oven or on a heating plate for 30 min. Check under a microscope to make sure that no more intact cells remained at the surface of the wells.&#160;In case the cells did not lyse completely, increase the heating time or add detergent. Freeze them once at -20 &#176;C also enhances breaking cells.</li>
		<li>Add 50 &#181;l neutralizing solution (40 mM Tris-HC, pH 4.0) to each well. Transfer all lysates into U-form wells of a PCR plate and dry in a PCR cycler at 95 &#176;C for 10 to 30 min. Reconstitute the lysates with 20 &#181;l distilled water.</li>
	</ol>
	</li>
	<li>Desalt using Drop-Dialysis9
	<ol>
		<li>use membrane filters with&#160;mean pore size 0.025 &#181;m.</li>
		<li>Float the filter on distilled water with shiny side up in wells of a 12-well plate. Wet the filter for 5 min. Pay attention to avoid air bubbles between the filter and water.</li>
		<li>Pipet the reconstituted lysates of each sample from step 3.1.4 carefully onto on each of the 4 areas of the filter. Cover the plate and dialyze for 30 to 60 min.</li>
		<li>Suck away the water under the filter without flipping over the filter nor disturbing the drops.</li>
		<li>Transfer the lysate-drops into wells of a new PCR-plate. Dry the DNA-drops by heating at 95 &#176;C for 10 to 30 min in a PCR cycler. Reconstitute the lysates in 10 &#181;l water.</li>
	</ol>
	</li>
</ol>
4. Digital PCR
<ol>
	<li>Thaw frozen components such as DNA, buffer and the assay mix at RT, spin down for 10 sec at maximum force of any available centrifuge (for example, VWR mini centrifuge) and then keep them on ice.</li>
	<li>Prepare master mixture containing all components except DNA (Table 1). Consider dead volume and calculate master mixture approximately 10% more than the required amount. 
	NOTE: Use the primers and probes used in the commercial kit.</li>
	<li>Vortex the master mixture, spin down for 10 sec at maximum force of any available centrifuge (for example, VWR mini centrifuge) and dispense 15 &#181;l to each well of a 96-PCR plate. Add all lysate (which contains DNA) of each sample to the wells containing the master mixture. Transfer 20 &#181;l of each reaction mixture into a well on the sample side of a cartridge at RT.</li>
	<li>Dispense 70 &#181;l of droplet generator oil into each well on the oil side of a cartridge. Close the cartridge with the gasket and place the whole setting into a droplet generator. Start droplet generation.</li>
	<li>Transfer 40 &#181;l droplet-solution into wells of a 96-well PCR plate. Seal the plate with a foil and place the plate into a PCR cycler. Set the lid-temperature at 105 &#176;C and ramp rate at 2 &#176;C/sec. Carry out PCR using the settings&#160;in Table 2. After the PCR, transfer the plate into a droplet reader and start reading. Alternatively, store the plate at 4 &#176;C for up to 24 hr.</li>
</ol>
5. Calculating Proportion of Tumor Cells
<ol>
	<li>Load the digital PCR data and perform automatic analysis.</li>
	<li>Control the results of each assay manually by inspecting the one-dimensional and 2-dimensional distribution of the positive and negative droplets, and sure that they are well separated and the threshold lines were in the right positions. Exclude results not meeting the criteria, for example, no clear separation of positive and negative droplets, from further analysis. Copy the resulting ratio NF1/RPP30 into an excel sheet.</li>
	<li>Calculate the proportion of tumor cells as: 2 x&#160;(1- NF1/RPP30).</li>
</ol>
6. Dose-dependent Change of Proportion of Tumor Cells
<ol>
	<li>For each drug-concentration, calculate the mean and standard deviation from the 4 replicates. Plot the means and the standard deviations against drug-concentrations.</li>
</ol>

<ol>
	<li>Caponigro, G., Sellers, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+preclinical+testing+of+cancer+therapeutic+hypotheses.">Advances in the preclinical testing of cancer therapeutic hypotheses.</a> Nat Rev Drug Discov. 10 (3), 179-187 (2011).</li><li>McMillin, D. W., Negri, J. M., Mitsiades, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+tumour-stromal+interactions+in+modifying+drug+response:+challenges+and+opportunities.">The role of tumour-stromal interactions in modifying drug response: challenges and opportunities.</a> Nat Rev Drug Discov. 12 (3), 217-228 (2013).</li><li>Wilding, J. L., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+for+drug+discovery+and+development.">Cancer cell lines for drug discovery and development.</a> Cancer Res. 74 (9), 2377-2384 (2014).</li><li>Edwards, A. 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=Preclinical+target+validation+using+patient-derived+cells.">Preclinical target validation using patient-derived cells.</a> Nat Rev Drug Discov. 14 (3), 149-150 (2015).</li><li>Frahm, 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=Genetic+and+phenotypic+characterization+of+tumor+cells+derived+from+malignant+peripheral+nerve+sheath+tumors+of+neurofibromatosis+type+1+patients.">Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve sheath tumors of neurofibromatosis type 1 patients.</a> Neurobiol Dis. 16 (1), 85-91 (2004).</li><li>Jiang, W., 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=Efficacy+and+selectivity+of+nilotinib+on+NF1-associated+tumors+in+vitro.">Efficacy and selectivity of nilotinib on NF1-associated tumors in vitro.</a> J Neurooncol. 116 (2), 231-236 (2014).</li><li>Jiang, W., Mautner, V. F., Friedrich, R. E., Kluwe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+assessment+of+the+anticancer+drug+response+of+plexiform+neurofibroma+tissue+using+primary+cultures.">Preclinical assessment of the anticancer drug response of plexiform neurofibroma tissue using primary cultures.</a> J Clin Neurol. 11 (2), 172-177 (2015).</li><li>Montero-Pau, J., Gomez, A., Munoz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+an+inexpensive+and+high-throughput+genomic+DNA+extraction+method+for+the+molecular+ecology+of+zooplanktonic+diapausing+eggs.">Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs.</a> Limnology and Oceanography-Methods. 6, 218-222 (2008).</li><li>Saraswat, M., Grand, R. S., Patrick, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desalting+DNA+by+drop+dialysis+increases+library+size+upon+transformation.">Desalting DNA by drop dialysis increases library size upon transformation.</a> Biosci Biotechnol Biochem. 77 (2), 402-404 (2013).</li><li>Iglesias-Bartolome, R., Martin, D., Gutkind, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+head+and+neck+cancer+oncogenome:+widespread+PI3K-mTOR+pathway+alterations+and+novel+molecular+targets.">Exploiting the head and neck cancer oncogenome: widespread PI3K-mTOR pathway alterations and novel molecular targets.</a> Cancer Discov. 3, 722-725 (2013).</li></ol>

The procedure is patent-protected (10 2013 221 452, Germany)

The key step in this genetic- and cell-based tool for assessing drug specificity in vitro is the determination of proportion of tumor cells using one or more tumor-specific genetic features. In contrast to conventional phenotypic features7, genetic features are specific, stable and easy to quantify. For example, a tumor-specific mutation or copy number variation is present exclusively in the tumor cells but not in non-tumor cells. Such a genetic stigma can be reliably and precisely quantified, for example, using digital PCR as demonstrated in this study.
For digital PCR, high purity of DNA is not essential. However, desalting and removing inhibitory molecules are necessary which can be achieved by a drop-dialysis using a suitable filter. The combined lysis and drop-dialysis is simple and quick, requires no special instruments and therefore can be carried out in any standard laboratory.&#160;If the cells do not break well, additional treatment can be considered such as freezing the cells at -20 &#176;C, adding detergent or/and using protease.&#160;
In the present study, the heterozygous deletion of the NF1 gene was used as the tumor-specific feature for the quantification. Similarly, deletions of other genes or loci can also be used. Furthermore, mutations can&#160;also be used for quantifying tumor cells. In such case, digital PCR assays for the mutant and the normal alleles need to be extensively validated to ensure their specificity for each of them.
Dose-dependent proportion of tumor cells provides an indication for drug-specificity or selectivity. Furthermore, it also enables extracting response of tumor cells from the response of total cells of a mixed culture to a drug (Figure 4). This extraction strategy provides a solution for the technical problem in using primary cultures (which contains both tumor and non-tumor cells) for drug-testing.
The genetic- and cell-based in vitro tool demonstrated in the present study may therefore have application potential (1) in drug-discovery where it provides a platform for assessing drug specificity or selectivity in vitro and (2) in personalized cancer care where it enables testing drugs in primary cultures and consequently may contribute to drug-selection in adjuvant chemotherapy4. Furthermore, pharmacological mechanism of a drug can be studied using this tool, since certain genetic alteration or multiple genetic alterations can be followed during the drug-treatment.
A difficulty in using individualized primary cultures is the lack a universal genetic feature for the quantification of the tumor cells. However, with today&#39;s advance in whole-genome sequencing, the most frequently altered genes and regions are known for common tumors. For example, among head and neck tumors, 71%, 23% and 22% have mutations in the TP53, FAT1 and CDKN2A genes, and 60%, 34% and 26% have deletions in the gene regions of CDKN2A, STK11 and PTEN, respectively10. Screening 5 to 10 such genes or/and regions by means of targeted-sequencing or/and digital PCR will therefore likely identify one or more genetic alterations which can be used for the quantification of tumor cells in the derived primary culture. Another limitation is the often insufficient amount of resected tumors for setting up primary cultures.
Despite the advantageous features and promising application potential of the genetic- and cell-based in vitro tool, its limitations should be kept in mind and its in vitro nature should be emphasized. For example, the differential effects or cytotoxicity of a drug on tumor cells may rather due to difference in cell types (fibroblasts vs. Schwann cells) or their different origins (from different individuals). The stronger effect of a drug on tumor cells than on non-tumor cells may also be explained by the fact that the former grow faster than the latter. Most importantly, cells in vitro behavior differs than cells in the human body and consequently, results of in vitro assessment often do not or only partially reflect the real clinical situation. Therefore, efficacy, cytotoxicity and specificity of drugs obtained for cultured cells are rather of biomarker nature but do not have the reliability of preclinically measured efficacy of antibiotics. Nevertheless, moving from cell lines to mixed cultures and/or primary cultures is a step forward in assessing drug-effect and specificity/selectivity in vitro. With extensive efforts, in vitro conditions may be found which enables reasonable correlation of in vitro data with real responses of patients, at least for some drugs.

Therapeutic effects of anticancer drugs on tumor cells are associated with various unwished toxic effects on non-tumor cells. Toxic effects are difficult to assess in the laboratory, largely due to the lack of suitable tools1. Cell cultures containing both tumor and non-tumor cells would provide a potential in vitro resource for assessing specificity or selectivity of a drug which may shed light on clinical toxicity. However, conventional in vitro assays such as proliferation or viability assays measure effect of a drug on total cells, but cannot divide the effect into the part on tumor cells and the part on non-tumor cells. For example, a 50% reduction of total cell number in a culture composed of 50% tumor cells and 50% non-tumor cells can imply death of all tumor cells, death of all non-tumor cells or, more likely, death of parts of both at various ratios. 
Also because of this technical problem, primary cultures which contain tumor and non-tumor stromal cells are frequently unsuitable for testing drugs. On the other side, because of this cellular heterogeneity, primary cultures resemble their original tumors far better than clonal cells in established cell lines2. More importantly, primary cultures are patient-specific and may enable individualized drug testing2-4.
An essential issue is therefore the development of a method for determination of the proportion of tumor cells in mixed cultures including primary cultures. Toward this challenge, a genetic solution was conceived which is based on determination of the quantitative ratio of a tumor-specific genetic feature to a universal reference sequence. From this ratio, the proportion of tumor cells in a mixed culture can be calculated. 
This study illustrates the procedure of the genetic and cell-based tool for assessing specificity of anticancer drugs including (1) preparing a mixed culture from tumor cells of an established line and non-tumor fibroblasts, (2) treating the mixed culture with a test drug, (3) preparing DNA for digital PCR, (4) determining the ratio of an tumor-specific heterozygous deletion to a reference sequence using a dual-probe droplet digital PCR assay, (5) calculating proportions of tumor cells at each drug concentration and (6) drawing up dose-dependent change of proportion of tumor cells.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">membrane filter of 25 mm diameter</td>
			<td style="width:71px;">Merk-Millipore</td>
			<td style="width:119px;">VSWP 02500</td>
			<td style="width:167px;">for drop-dialysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ddPCR assay for NF1</td>
			<td style="width:71px;">BioRad</td>
			<td>dHsaCP1000177</td>
			<td>FAM-labelled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ddPCR assay for RPP30</td>
			<td style="width:71px;">BioRad</td>
			<td>dHsaCP2500350</td>
			<td>HEX-labelled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QX200</td>
			<td style="width:71px;">BioRad</td>
			<td style="width:119px;">186-4001</td>
			<td>the previous version QX was used in the study, which is not available any more. The replacement is QX200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Droplet oil</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cartriges&#160;</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gaskets</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cartrige holder</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pierceble foil</td>
			<td style="width:71px;">BioRad</td>
			<td>181-4040</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing specificity of anticancer drugs in vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Quality of DNA prepared using the combined HotShot/Drop-Dialysis is sufficient for satisfactory digital PCR (Figure 1B). For 2,000 fibroblasts, approximately 3,000 &#177; 500 positive droplets were obtained, corresponding to 75% of the expected 4,000 copies (two copies in one cell). In this study, the tumor cells were from an established line MPNST S462 which are known to have a heterozygotic deletion of the NF1 gene5. This 50% gene dosage reduction was clearly detected by the dual digital PCR assay (Figure&#160;1B). By contrast, two copies of this gene were measured in fibroblasts. This genetic difference between tumor and non-tumor cells enables calculation of the proportion of tumor cells in mixed cultures (Figure&#160;2). By measuring the relative gene dosage of NF1 to RPP30, proportion of tumor cells in a mixed culture can be determined at each drug-concentration (Figure&#160;3).
<img alt="Figure 1" src="/files/ftp_upload/53752/53752fig1.jpg" /> 
Figure 1. Digital PCR.&#160;(A) illustration of the principle. (B) a dual-probe assay reveals reduced amount of a target (NF1) gene in the tumor cells MPNST S462 in relation to a reference (RPP30) gene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53752/53752fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53752/53752fig2.jpg" /> 
Figure 2. Calculated Proportion of Tumor Cells. Tumor cells and non-tumor cells were mixed at various ratios and seeded into each well of a 96-plate. After attachment O/N, the cells were lysed with HotShot/Drop-dialysis and the desalted lysates were subjected to digital PCR which gave the ratio of NF1/RPP30. Base on this ratio, proportion of tumor cells in each well was calculated. Mean and standard deviation of proportion of tumor cells were calculated from 4 replicates at each set-up proportion. The calculated proportions of tumor cells (Y-axis) were plotted against the set-up proportions (X-axis). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53752/53752fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53752/53752fig3.jpg" /> 
Figure 3. Dose-dependent Change of Proportion of Tumor Cells.&#160;(A) dose-dependent reduction of visible vital cells. (B) proportion of tumor cells (mean and standard deviation of 4 replicates) calculated from the ratio of NF1: RPP30, decreased in the range of 0 - 10 &#181;M. At the highest dose 50 &#181;M, few vital cells were left, likely due to toxic effect to all cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53752/53752fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53752/53752fig4.jpg" /> 
Figure 4. Separating Response of Tumor Cells from That of Non-tumor Cells.&#160;(A) viability or/and proliferation of total cells can be measured using conventional assays; Proportion of tumor cells can be determined using the procedure demonstrated in the present study. (B) using the two parameters, response of total cells in a mixed culture to a drug can be divided into response of the tumor cells and response of the non-tumor cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53752/53752fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:147px;"></td>
			<td style="width:171px;">1 Reaction</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:147px;">2 x ddPCR supermix</td>
			<td style="width:171px;">12</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:147px;">Assay mix for NF1</td>
			<td style="width:171px;">1.2</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:147px;">Assay mix for RPP30</td>
			<td style="width:171px;">1.2</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:147px;">HaeIII</td>
			<td style="width:171px;">0.6</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:147px;">Subtotal</td>
			<td style="width:171px;">15</td>
			<td style="width:99px;">Master mixture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:147px;">DNA</td>
			<td style="width:171px;">9</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:147px;">Total</td>
			<td style="width:171px;">24</td>
			<td style="width:99px;">Final PCR Mixture</td>
		</tr>
	</tbody>
</table>
Table 1 Setting Up Dual ddPCR Reaction
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Action</td>
			<td>Temperature</td>
			<td>Time</td>
			<td>Cycles</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Polymerase activation</td>
			<td>95 &#176;C</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>2</td>
			<td>DNA denaturation</td>
			<td>94 &#176;C</td>
			<td>30 sec</td>
			<td rowspan="2">40</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Annealing/exetension</td>
			<td>60 &#176;C</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Polymerase deactivation</td>
			<td>98 &#176;C</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Hold (optional)</td>
			<td>4 &#176;C</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2 PCR Cycler Settings

Watch this Scientific Journal Video about Assessing Specificity of Anticancer Drugs In Vitro at JoVE.com

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

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9.4K Views.  University Medical Center Hamburg-Eppendorf. The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

Video: Assessing Specificity of Anticancer Drugs In Vitro

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...