The authors acknowledge the Institute for Mental and Physical Health and Clinical Translation (IMPACT) SEED funding, the &#34;Alfred Deakin Postdoctoral Research Fellowship&#34; program at Deakin University, and the &#34;Australian Government Research Training Program Scholarship&#34;.&#160;

NOTE: Prior to starting the experiment, wear personal protective equipment, including a lab coat, gloves, and goggles. See the Table of Materials for details about materials, reagents, equipment, and software used in this protocol.
1. Buffers required for the assay
<ol>
	<li>Prepare the buffers required for this experiment-the SELEX buffer required for aptamer folding, Blocking Buffer (BB), Wash Buffer (WB), and Binding Buffer (BiB) (Table 1)-freshly on the day of the experiment and keep them on ice or at 4 &#176;C. 
	NOTE: Each aptamer requires a unique folding condition. This includes the SELEX buffer and folding temperature conditions. Care should be taken to fully replicate the methods from the original paper describing the aptamer10. In this experiment, all buffers are prepared in Dulbecco&#39;s phosphate-buffered saline (DPBS). The buffer volume required in each experiment depends on the number of cell lines, number of replicates, and number of aptamer concentrations that are tested.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td colspan="2">Ingredients</td>
			<td rowspan="2">Volume required</td>
		</tr>
		<tr>
			<td>Item</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>SELEX buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td>50 &#181;L per sample + 10% pipetting error</td>
		</tr>
		<tr>
			<td rowspan="4">Blocking Buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td rowspan="4">500 &#181;L per cell line</td>
		</tr>
		<tr>
			<td>BSA a</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td>tRNA b</td>
			<td>0.1 mg/mL</td>
		</tr>
		<tr>
			<td>FBS c</td>
			<td>10% (v/v)</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td>1 mL for the first wash + 100 &#181;L per test sample + 10% pipetting error</td>
		</tr>
		<tr>
			<td rowspan="4">Binding Buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td rowspan="4">50 &#181;L per sample + 10% pipetting error</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>2 mg/mL</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>20% (v/v)</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers required for the binding assay. aBovine Serum Albumin, bTransfer Ribonucleic Acid, cFetal Bovine Serum.
2. Preparation of aptamers
NOTE: The aptamers used in the assay are tagged with a fluorescence reporter molecule, and therefore care should be taken to protect them from light.
<ol>
	<li>Prior to the experiment, prepare a 100 &#181;M stock (stock A) of test and control aptamers using pyrogen- and RNase-free ultrapure water (Figure 1). 
	NOTE: For long-term preservation, stock A should be kept in a freezer at -20 &#176;C.</li>
	<li>Prepare stock B as the working concentration of aptamers by diluting stock A using SELEX buffer (Table 1). To follow this protocol, dilute stock A to a 1,000 nM stock to prepare stock B (Figure 1).</li>
	<li>To make the aptamer ready for the formation of the 3-dimensional (3D) structure, in a 250 &#181;L tube, dilute stock B with SELEX buffer to prepare the required volume and concentration of the aptamer for folding. 
	NOTE: The folded aptamers will be exposed to an equal volume of cells. Therefore, the concentration of the aptamer that is set for folding should be 2x more concentrated than the desired final concentration. Use equation (1) to calculate the required volumes and concentrations. Remember to consider an extra 10% volume for the pipetting error. 
	Concentrationstock A &#215; Volumestock A = Concentrationstock B &#215; Volumestock B&#160; (1)</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64304/64304fig01.jpg" /> 
Figure 1: A diagram showing the steps in the preparation of aptamers. 1Stock 1 is stored at -20 &#176;C for long-term preservation. 2Working concentrations are prepared in SELEX buffer and are not stored. <a href="https://www.jove.com/files/ftp_upload/64304/64304fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Maintenance of cancer cells
NOTE: Prior to commencement of the study, make sure that the cells are at their early passage numbers, show their typical morphological features, and are mycoplasma free. To test the selectivity and specificity of the aptamer, cell lines that are high, moderate, and low/negative expressors of the protein of interest are ideally required.
<ol>
	<li>Seed the cells in a T75 culture flask, using appropriate culture conditions. Grow them in a 5% CO2 humidified incubator, at 37 &#176;C. 
	NOTE: In this study, Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (complete medium) was used.</li>
	<li>When the cells reach ~80% confluency, passage them to a new flask containing fresh complete medium. 
	NOTE: Depending on the protein of interest and the cell line, 80% confluency could provide a suitable cell population for the binding assay. For the cell lines in this experiment, MDA-MB-231 and HEK 293T, 80% confluency is suitable. At this stage, proceed to section 4, the binding assay. Always check the expression of the protein of interest, using mAbs specific for that protein.</li>
</ol>
4. Binding assay
NOTE: Figure 2 summarizes the steps required in the binding assay in adherent cells.
<ol>
	<li>In a class II biosafety cabinet, collect the cells of each flask in tubes as follows:
	<ol>
		<li>Collect and discard the media in the flask, add 2 mL of PBS, spread it over the cells, and then, collect and discard the PBS. Repeat this step twice more to remove all traces of media that may inactivate trypsin. Add 1 mL of 0.25% of trypsin/EDTA to each flask and incubate for 5-10 min at 37 oC. Visualize the detachment of cells under a microscope.</li>
		<li>Add 1 mL of complete medium to the cells, and pipette the cells up and down to make a single-cell suspension. Pipette the cells into an appropriate tube and centrifuge at 200 &#215; g for 5 min. 
		NOTE: For non-adherent cells, collect the cells in a tube, centrifuge (200 &#215; g, 5 min), and proceed to step 4.1.3.</li>
		<li>Discard the supernatant and resuspend the cells in 1 mL of fresh medium. Count the cells using trypan blue staining, by diluting a certain volume of cell suspension with trypan blue. Distribute ~15 &#181;L of the mixture between a hemocytometer and a cover glass. Count the cells as previously described18, using equation (2): 
		<img alt="Equation 1" src="/files/ftp_upload/64304/64304eq01.jpg" style="margin: auto;" /> (2) 
		NOTE: Use the minimum possible volume of cell suspension and take a note of the dilution factor. For example, mixing equal volumes of cell suspension and 0.04% trypan blue gives a dilution factor of 2. Ensure high viability (live cells/total cells &#215; 100) of ~90% for most adherent cell lines before proceeding. Dead cells non-specifically take up aptamers and alter the results19. It is possible to use other cell counting techniques, such as using a cell counter.</li>
		<li>Collect the required number of cells, making sure to have 10 &#215; 104 cells per test sample. Consider an extra 10% volume for pipetting error. 
		NOTE: It is important to always keep the same cell count between experiments and replicates.</li>
		<li>Incubate the cells at 37 &#176;C for 2 h to allow for the stabilization of the protein of interest on the cell membrane following enzymatic detachment. 
		NOTE: This incubation period might differ according to the protein of interest.</li>
	</ol>
	</li>
	<li>During this 2 h incubation:
	<ol>
		<li>Set the temperature of the centrifuge to 4 &#176;C. Leave tRNA and stocks of aptamer at room temperature or on ice to thaw. To protect the fluorescence reporter molecule, protect the aptamer tubes from light. 
		NOTE: The role of tRNA is to block the nucleic acid binding sites.</li>
		<li>Prepare the SELEX buffer, BB, WB, and BiB (see section 1), keeping them all on ice or at 4 &#176;C. Set the thermocycler machine on an empty cycle. Place a 96-well black plate and flow cytometry tubes on ice. 
		NOTE: Setting the thermocycler on an empty cycle prepares the cooling and heating system and helps generate more reproducible results.</li>
	</ol>
	</li>
	<li>Following the 2 h incubation, centrifuge the cells at 500 &#215; g for 5 min. Discard the supernatant and resuspend the cells in 500 &#181;L of BB. Incubate the cells at 4 &#176;C for 30 min with intermittent mixing.</li>
	<li>During this 30 min incubation, perform aptamer folding as follows:
	<ol>
		<li>Make up the 2x concentrations of aptamers (see section 2), and then mix and incubate the aptamers in the thermocycler machine, according to the required folding conditions. For this EpCAM aptamer, use the following folding conditions of 95 &#176;C, 5 min, followed by 22 &#176;C, 10 min, and 37 &#176;C, 15 min. 
		NOTE: Always include a negative control (i.e., SELEX buffer without aptamers).</li>
	</ol>
	</li>
	<li>Following the 30 min incubation, centrifuge the cells (500 &#215; g, 5 min, 4 &#176;C), remove the supernatant, add 1 mL of WB, and centrifuge the cells again (500 &#215; g, 5 min, 4 &#176;C). Remove the supernatant and resuspend the cells in a suitable volume of the BiB.</li>
	<li>Pipette 50 &#181;L of the resuspended cells into each well of an ice-cold, 96-well black plate. Keep the cells on ice to inhibit internalization of the protein of interest.</li>
	<li>Pipette 50 &#181;L of the aptamers onto a 50 &#181;L volume of cells, mix, and incubate in darkness at 4 &#176;C for 30 min. Centrifuge the plate at 500 &#215; g, 5 min, 4 &#176;C, and carefully remove the supernatant.</li>
	<li>Carefully resuspend the pellet in WB and centrifuge at 500 &#215; g for 5 min. Repeat the wash step (4.7) 2x and resuspend in 100 &#181;L of WB for flow cytometric analysis. 
	NOTE: See Figure 3 for a diagram of interactions between aptamers and cells.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64304/64304fig02.jpg" /> 
Figure 2: A diagram depicting the steps in performing an aptamer-protein-binding assay. Abbreviations: SELEX = Systematic Evolution of Ligands by EXponential Enrichment; BB = Blocking Buffer; WB = Wash Buffer; BiB = Binding Buffer. <a href="https://www.jove.com/files/ftp_upload/64304/64304fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64304/64304fig03.jpg" /> 
Figure 3: A diagram showing the different types of cells and aptamers required to perform the aptamer binding assay. Abbreviation: EpCAM = epithelial cellular adhesion molecule. This figure was created using Biorender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64304/64304fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Flow cytometry and data analysis
NOTE: Before turning on the flow cytometer, make sure that there are no &#34;bubbles&#34; in the membrane filter units for the shut-down solution, cleaning solution, and sheath fluid (0.9% NaCl). &#34;Bleed out&#34; bubbles if there are bubbles in the capsules. Make sure that the waste container is empty, and containers of sheath fluid, water, and 1% bleach in ultrapure water are full.
<ol>
	<li>Turn on the flow cytometer and then the computer. 
	NOTE: The details of running the flow cytometer explained here are specific to the machine and software demonstrated in the video (see Table of Materials). Other software would require appropriate training to use.</li>
	<li>Open the flow cytometry analysis software, log in to the program, and under the Cytometer tab, run Fluidics Start up.</li>
	<li>To create a new experiment, under the experiment tab, click New Folder and name the folder/experiment appropriately.</li>
	<li>Click on new folder to highlight, then under the experiment tab again, click New Experiment and name the experiment appropriately.</li>
	<li>To add the first sample/specimen, under the experiment tab, click New Specimen and name this specimen appropriately (name of cell line/control sample/ experiment sample).</li>
	<li>To add a tube sample, highlight the specimen (group) and under the experiment tab, click New Tube. Add the appropriate number of tubes and name.</li>
	<li>To prepare the required graphs, under the worksheet tab, open a new worksheet. Once the new worksheet window pops up, open the following using the worksheet screen (hover the mouse across the logo/pictures to find the names):
	<ol>
		<li>Prepare a dot blot graph of forward scatter (FSC) versus side scatter (SCC) to select the population of interest. Define the first gate by identifying and selecting the population of interest (P1) in a forward and side scatter density plot. Exclude the debris, which constitutes the population with the lowest forward scatter signal. 
		NOTE: The FSC parameter detects cells or events based on their size and the SCC discriminates them based on their granularity20.</li>
		<li>Prepare a dot blot graph of FSC-area (FSC-A) versus FSC-height (FSC-H) to select the single-cell population. Define the second gate by excluding doublet cell populations, as doublet cells considerably affect the results and conclusions. Exclude doublets by using FSC-H versus FSC-A density plots, where cells of the same size show a similar area and height. Hence, the singlets get clustered diagonally and separated from doublets. 
		NOTE: FSC is roughly proportional to the cell size. The voltage pulses are defined as FSC-H, the intensity of the signal, FSC-width that reflects cell size and the duration of the signal, and FSC-A, which is H &#215; W. Doublets have a double width and area value; therefore, gating for singlets is based on detecting disproportions between H, W, and A caused by doublets.</li>
		<li>Prepare a histogram of the number of events against the fluorophore of interest.</li>
	</ol>
	</li>
	<li>Before starting flow cytometry, ensure that the acquisition dashboard for controlling the sample acquisition, inspector, and cytometer to adjust voltage parameters, as well as the worksheet with all the graphs are open. 
	NOTE: At least 100 &#181;L of a 10 &#215; 104 cell suspension in a flow cytometry tube is needed to perform the analysis. Especially in case of lower viabilities, propidium iodide staining can be performed to select the viable cell population21,22.</li>
	<li>To run the first sample, on the left-hand side of the screen make sure the arrow pointing to the tube is green. If this arrow is not green, click on the arrow to make it green.</li>
	<li>Using a pipette, transfer each sample from the 96-well black plate to a flow cytometry tube. Run the untreated, unstained control sample on a low speed.</li>
	<li>On the acquisition dashboard, choose an appropriate number of events to record (30,000), change the flow rate to low, and click Acquire Data.</li>
	<li>Adjust the voltage for the FSC and SCC parameters. Ensure the cell population is centralized within the dot plot and that no cells are touching either axis of the graph to avoid losing the cells of interest.</li>
	<li>Increase the acquisition speed to medium or high to analyze the samples faster but do not exceed more than 200 events/s. Then, click Record Data.</li>
	<li>Perform the gating for P1 (Figure 4A) and the single-cell population (Figure 4B). Construct the histogram of events against the used fluorochrome and select P1 based on the data (allophycocyanin (APC) in this case) (Figure 4C).</li>
	<li>After adjusting the voltage, gating and recording the data, take out the sample and click Next Tube.</li>
	<li>Insert the next sample, and repeat recording data for all control and test samples (Figure 3).</li>
	<li>Once all the data are collected, wash the flow cytometer by running three tubes of 50% bleach, FACS rinse, and ultrapure water, each for 5 min at a high flow rate.</li>
	<li>Then, from the Cytometer dropdown menu, click Fluidics Shut Down.</li>
	<li>Prior to closing the software and turning off the machine and the computer, export the results as .fcs files to a USB drive to transfer and analyze them, as follows:
	<ol>
		<li>In the analysis software, press the NEW button to create a new document and window to handle the analysis. Drag the sample files into the new window.</li>
		<li>Double-click to open the unstained sample. Choose the P1 population, double-click on the P1 population to create a FSC-H versus FSC-A graph, and gate the single-cell population.</li>
		<li>Double-click on the gated single cells to create a histogram of events against the used fluorochrome.</li>
		<li>In the original window, select P1 and single cells and drag them to All Samples so that all samples now contain the same gating.</li>
		<li>Click on the Layout Editor button to open the Layouts window. Drag two samples (control and test) over one another to create an overlay histogram.</li>
	</ol>
	</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue Culture. , 395-397 (1973).</li><li>McKeague, 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=The+minimum+aptamer+publication+standards+(MAPS+guidelines)+for+de+novo+aptamer+selection.">The minimum aptamer publication standards (MAPS guidelines) for de novo aptamer selection.</a> Aptamers. 6, 10-18 (2022).</li><li>Schoofs, G., Van Hout, A., D'huys, T., Schols, D., Van Loy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+to+identify+compounds+that+disrupt+binding+of+fluorescently-labeled+CXC+Chemokine+ligand+12+to+CXC+Chemokine+receptor+4.">A flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled CXC Chemokine ligand 12 to CXC Chemokine receptor 4.</a> Journal of Visualized Experiments. (133), e57271 (2018).</li><li>McKinnon, 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=Flow+cytometry:+an+overview.">Flow cytometry: an overview.</a> Current Protocols in Immunology. 120 (1), 1-11 (2018).</li><li>Kamiloglu, S., Sari, G., Ozdal, T., Capanoglu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+cell+viability+assays.">Guidelines for cell viability assays.</a> Food Frontiers. 1 (3), 332-349 (2020).</li><li>Ruscito, A., DeRosa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+binding+aptamers:+Selection+strategies,+characterization,+and+applications.">Small-molecule binding aptamers: Selection strategies, characterization, and applications.</a> Frontiers in Chemistry. 4, 14 (2016).</li><li>McKeague, 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=Comprehensive+analytical+comparison+of+strategies+used+for+small+molecule+aptamer+evaluation.">Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation.</a> Analytical Chemistry. 87 (17), 8608-8612 (2015).</li><li>Henri, J., Bayat, N., Macdonald, J., Shigdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+using+nucleic+acid+aptamers+in+cell+based+assays.">A guide to using nucleic acid aptamers in cell based assays.</a> Aptamers. 23, (2019).</li><li>Mao, H., 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=The+mechanism+and+regularity+of+quenching+the+effect+of+bases+on+fluorophores:+the+base-quenched+probe+method.">The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method.</a> Analyst. 143 (14), 3292-3301 (2018).</li><li>McKeague, 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=Analysis+of+in+vitro+aptamer+selection+parameters.">Analysis of in vitro aptamer selection parameters.</a> Journal of Molecular Evolution. 81 (5), 150-161 (2015).</li><li>Chen, B., 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=Targeting+negative+surface+charges+of+cancer+cells+by+multifunctional+nanoprobes.">Targeting negative surface charges of cancer cells by multifunctional nanoprobes.</a> Theranostics. 6 (11), 1887 (2016).</li><li>Shigdar, 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=RNA+aptamers+targeting+cancer+stem+cell+marker+CD133.">RNA aptamers targeting cancer stem cell marker CD133.</a> Cancer Letters. 330 (1), 84-95 (2013).</li><li>Amraee, M., Oloomi, M., Yavari, A., Bouzari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+aptamer+identification+and+characterization+for+E.+coli+O157+detection+using+cell+based+SELEX+method.">DNA aptamer identification and characterization for E. coli O157 detection using cell based SELEX method.</a> Analytical Biochemistry. 536, 36-44 (2017).</li><li>Yu, X. -. X., 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=Selection+and+characterization+of+a+novel+DNA+aptamer,+Apt-07S+specific+to+hepatocellular+carcinoma+cells.">Selection and characterization of a novel DNA aptamer, Apt-07S specific to hepatocellular carcinoma cells.</a> Drug Design, Development and Therapy. 14, 1535 (2020).</li></ol>

The authors have no conflicts of interest to disclose.

The key challenge with developing new aptamers is the lack of standard guidelines that applies to different steps of this process. McKeague et al. have recently demonstrated some of the associated challenges, which lead to unclear presentations of data in publications and failure to replicate the research. They proposed fundamental guidelines necessary for consideration in characterizing aptamers19. An aptamer binding assay is a critical step in screening and/or characterizing aptamers<sup class="...

Cancer is still one of the leading causes of mortality worldwide1. Despite the significant improvement in cancer treatment in recent decades, anticancer drug development is still a highly debated topic. This is because chemotherapy, as the mainstay of cancer treatment, is accompanied by serious side effects that limit patient compliance with the treatment. Moreover, chemotherapy-induced cancer resistance to treatment has restricted its application as the sole choice of medical intervention. The application of monoclonal antibodies (mAbs) introduced an enhanced response to cancer treatments2. The rationale of using mAbs was to improve the efficacy of chemotherapeutics and minimize their adverse reactions. However, the administration of mAbs also became a challenge. This was not only because of the mAb-induced immunological reactions but also due to the animal-dependent and expensive production costs and difficult storage conditions3. Introduction of aptamers in the 1990s4 raised new hopes in cancer treatment, as the application of aptamers could address the challenges associated with mAbs.
Aptamers are short nucleic acid sequences that are specifically produced for a certain target. Systematic evolution of ligands by exponential enrichment (SELEX) is a common method in aptamer production. In SELEX, the protein of interest is incubated with a library of random nucleotide sequences, and through a series of iterative cycles, the aptamer specific for that protein is purified. Aptamers have similar target selectivity and specificity to mAbs, and therefore drug development in this field shows promising future applications. Aptamers specific for cancer biomarkers could be applied as single drugs and cancer diagnostic tools5,6,7. Due to their nano-sized structure, these aptamers could also act as drug carriers to deliver cytotoxic agents specifically to the tumor8. This would increase the efficacy of targeted drug delivery and decrease chemotherapy-associated, off-target adverse reactions. Moreover, these nanomedicines have a high tissue penetration, which makes them a desirable candidate for deep-tumor drug delivery and treatment. Aptamers can also be designed to target the transporters expressed on the blood-brain barrier (BBB) to improve drug delivery to brain tumors9. A good example of such an aptamer are bifunctional aptamers, targeting the transferrin receptor (TfR)10 to enhance drug delivery across the BBB, and delivering a cytotoxic drug payload to tumor cells11.
Despite all the advantages of aptamers, drug development in this field has not yet yielded a marketed, successful anticancer drug. One reason for this could be the lack of standard and reproducible methods that could be followed globally by researchers in the field. In this paper, a step-by-step protocol of an aptamer binding to a native protein expressed on the cell surface is demonstrated. This protocol is a prerequisite step in the preclinical assessment of anticancer aptamers. The assay is performed to show the selectivity and specificity of the purified aptamer collected from SELEX or a published aptamer sequence for confirmation of selectivity and specificity. This flow cytometric-based assay is a rapid, reliable, sensitive assay that accurately shows the selectivity and specificity of the aptamer, where the aptamer is being tested against proteins on the cell surface12,13,14. This method is demonstrated using the binding of an aptamer specific for EpCAM shown in this paper15. EpCAM, as a transmembrane glycoprotein, plays roles in tumor cell signaling, progression, migration, and metastasis16,17. To show the selectivity and specificity of this aptamer, EpCAM-positive and -negative cancer cells were used. The previously developed EpCAM specific aptamer, TEPP (5&#8242;-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3&#8242;), and a negative control aptamer, TENN (5&#8242;-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3), were used as EpCAM-binding and -non-binding aptamers, respectively10. The 3&#39; end of both TEPP and TENN were labeled with a TYE665 fluorophore.
TEPP is a bifunctional aptamer that targets EpCAM from one end and TfR on the other. This has made TEPP a suitable candidate for drug delivery to EpCAM+ brain tumors. Using its TfR-specific end, TEPP traverses the blood-brain barrier, and using the EpCAM-specific end, finds the tumor and delivers its cargo (e.g., cytotoxic drugs) to the tumor. TENN has a similar length and 2D structure as TEPP, but it does not have affinity for the EpCAM or TfR, and hence is a suitable negative control aptamer. Using TEPP and TENN, testing the binding of an aptamer to the target protein using flow cytometry is shown in this paper. This protocol applies to the development of cell-specific aptamers. It is also applicable to further complementary and confirmation analyses of the aptamer sequences available in the literature. The protocol can also be used by those new to the aptamer field who are looking at using a previously published aptamer for their research and development (R&#38;D) purposes. In this paper, two aptamer sequences available in the literature are studied.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes with attached lid</td>
			<td>Sigma-Aldrich</td>
			<td>T6649</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL CellStar blue screw cap, conical bottom tube</td>
			<td>Greiner Bio One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipettes</td>
			<td>Greiner Bio One</td>
			<td>606180</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Becton Dickinson Cytometer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva V9.0</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Lyophilized powder</td>
			<td>Sigma-AldrichTM</td>
			<td>A7906-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-line Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM) High Glucose Media Powder</td>
			<td>Life Technologies</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate- Buffered Saline (DPBS)</td>
			<td>Life Technologies</td>
			<td>21300025</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo, LLC 10.8.1</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS)</td>
			<td>Bovogen</td>
			<td>SFBS-F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>American Type Culture Collection</td>
			<td>ACS-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>American Type Culture Collection</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, PS, 96 well, F-bottom (Chimney well), Black</td>
			<td>Greiner Bio One</td>
			<td>655076</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniAmp Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) tablets</td>
			<td>Life Technologies</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrogen- and RNase-free ultrapure water</td>
			<td>Milli-Q</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Cell Culture flask</td>
			<td>Cellstar</td>
			<td>658170</td>
			<td></td>
		</tr>
		<tr>
			<td>TENN</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td>5&#8242;-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3</td>
		</tr>
		<tr>
			<td>TEPP</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td>5&#8242;-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3&#8242;</td>
		</tr>
		<tr>
			<td>Transfer RNA (tRNA)</td>
			<td>Sigma-Aldrich</td>
			<td>R8508-5X1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based cell surface protein binding assay for assessing selectivity and specificity of an anticancer aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

An important aspect of new drug discovery and development is assuring the selectivity and specificity of the drug candidate. This means that the drug candidate should be able to discriminate between different cells and only affect the cell population of interest (selectivity). Selectivity is studied using cell lines that differ in terms of expression of the protein of interest. In this study, MDA-MB-231 and HEK 293T cell lines were chosen as EpCAM-positive and -negative cells. Specificity is another determinant that show...

Watch this Scientific Journal Video about A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer at JoVE.com

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

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JoVE Journal

Cancer Research

3.2K Views.  Deakin University. A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein. 

Video: A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...