We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.

1. Cleaning the Glass Surface and Preparing Reagents
<ol>
	<li>Place a glass surface in an oxygen plasma machine for 5 min at 50% power to clean it.</li>
	<li>Prepare 2.5 ml 2% reconstituted (3-aminopropyl) triethoxysilane (APTES) solution, by adding 50 &#181;l of APTES and 2.45 ml of ethanol in a conical tube.</li>
</ol>
2. APTES and DSB Functionalization
<ol>
	<li>Add APTES solution to the surfaces. Pipette 150 &#181;l per well for 8 well plates. Pipette 100 &#181;l per well for 24 well plates. Pipette 1.1 ml for 60 x 15 mm glass dishes. Cover the surfaces to prevent evaporation and uneven distribution of APTES solution. Place the surfaces on a platform shaker for 50 min at room temperature, which creates an even distribution of the APTES layer. 
	NOTE: APTES is an aminosilane that forms the first layer of the surface. If using a different surface, heuristically determine the volume of solution needed to cover the surface.</li>
	<li>Select the temperature for the oven, while the surfaces are on the shaker: Heat to 55 &#176;C for 2 hr for the 8 well plates and 24 well plates. Heat glass only dishes to 90 &#176;C for 1 hr.</li>
	<li>Rinse the surfaces with ethanol.
	<ol>
		<li>Administer the amount of ethanol required by referencing based on the surface used. Add 150 &#181;l ethanol to each well for 8 well plates. Add 125 &#181;l ethanol to each well for 24 well plates. Add 1.1 ml ethanol for glass dishes.</li>
		<li>Rinse the surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface. Rinse twice more using ethanol. 
		NOTE: If any of the glass bottom well plates have any plastic in them, do not heat them above 65 &#176;C, as the plastic will begin to melt and warp.</li>
	</ol>
	</li>
	<li>Dry with 100% nitrogen gas dispensed from a tank. Place the surfaces in the oven.</li>
	<li>Prepare 2.5 ml of d-desthiobiotin (DSB) solution by combining 1.5 mg/ml DSB in 37.5 &#181;l of dimethyl sulfoxide (DMSO), and 5 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in 2462.5 &#181;l of the 0.1 M 4-morpholinoethanesulfonic acid hydrate (MES) (pH 6) buffer. Then combine both solutions.</li>
	<li>Add 1 &#181;l of 2-&#946; mercaptoethanol, after 15 min, into the solution to quench the reaction between DSB and EDC. Remove hot APTES functionalized glass surfaces from the oven. Wait 5-10 min for the glass surfaces to cool.</li>
	<li>Add MES buffer the surface to rinse, using amounts based on the surface. Add 150 &#181;l MES buffer to each well for 8 well plates. Add 125 &#181;l MES buffer to each well for 24 well plates. Add 1.1 ml MES buffer for glass dishes.</li>
	<li>Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface. Rinse twice more with MES buffer.</li>
	<li>Apply the DSB solution to the surfaces to allow them to incubate. Add 150 &#181;l DSB solution per well for 8 well plates. Add 100 &#181;l DSB solution per well for 24 well plates. Add 1.1 ml for glass dishes DSB solution.</li>
	<li>Place the DSB covered glass surfaces on a damp paper towel inside a Petri dish. Cover and incubate in a 4 &#176;C fridge for 18-24 hr.</li>
</ol>
3. Streptavidin Functionalization
<ol>
	<li>Rinse each glass surface three times with 1 ml of 1.0x Phosphate Buffered Saline (PBS). Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface. Dilute the streptavidin (SAv) stock solution to 0.4 mg/ml (recommended). 
	NOTE: PBS is isotonic so it can be used as a medium for dilution and rinsing. PBS is used as a solvent for the SAv, and therefore, will not affect the ability of the SAv to bind to the surface.</li>
	<li>Apply 0.4 mg/ml of SAv solution evenly to the surfaces so that a thin layer of forms at the bottom of the glass, select the amount of solution based on surface. For 8 well plates, add 150 &#181;l 0.4 mg/ml SAv solution per well. For 24 well plates, add 100 &#181;l 0.4 mg/ml SAv solution per well. For glass dishes, add 1.1 ml 0.4 mg/ml SAv solution.</li>
	<li>Cover and move the plates to a 14 cm Petri dish to retain moisture. Incubate the Petri dish in refrigerator for 18-24 hr. 
	NOTE: It is essential to utilize consistent volumes of APTES, DSB, and SAv on each surface.</li>
	<li>Rinse each glass surface three times with 150&#160;&#181;l of PBS to remove SAv. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface.</li>
	<li>Wet a paper towel with de-ionized water and place the paper towel flat in 14 cm Petri dish surrounding the plates to retain moisture in the wells. Cover the Petri dish containing the wells. Incubate the Petri dish in a 4 &#176;C Biosafety level 1 (BSL-1) refrigerator until needed.</li>
</ol>
4. Cell Capture and Release
<ol>
	<li>Start with T175 flask(s) of cells intended for the experiment. Aspirate the media from flask(s). Wash out the remaining media with 5 ml of room temperature PBS. Aspirate PBS from the flask(s).</li>
	<li>Add 10 ml of non-enzymatic lifting agent, such as Cell Dissociation Solution, to the T-175 flask of cells. Put the flask in the incubator for 6 min to allow for lifting of the cells from the flask.</li>
	<li>After 6 min, add 10 ml of cold Hank&#39;s Balanced Salt Solution (HBSS; See List of Materials) to inactivate the lifting agent. Take out 20 &#181;l of cells to count number of cells in solution using a hemacytometer. 
	NOTE: Depending on the functionalized surfaces used in the capture experiment, the number of cells needed varies. For functionalized 8 well plates, use 300,000 cells per well. For functionalized glass dishes, use 1.1 million cells. For functionalized 24 well plates, 125,000 cells are recommended. More information on cell counting is found in the Supplement.</li>
	<li>Repeat steps 4.1- 4.3 for another flask of cells, if fewer cells are present than needed for the experiment. Combine cell suspensions and recount cells to get total number of cells in solution.</li>
	<li>Spin down cell suspension in a centrifuge at 500 x g for 5 min at 4 &#176;C to get a pellet of concentrated cells. Find the appropriate amount of HBSS to add to the cell suspension to get a concentration of 1 x 106 cells per ml, then aspirate the supernatant from the spun down cells, and add the calculated volume. This volume can be calculated using the equations in the Supplement.</li>
	<li>Pipette up and down (triturate) to resuspend the cells in solution and reduce cellular clumping in solution that may reduce antibody binding. Split the cellular solution into separate control and experimental solutions. View Supplemental File for components and calculations.</li>
	<li>Add the biotinylated antibodies to the respective cell solutions as described in the Supplemental File. Incubate for 30 min at 4 &#176;C on an end-over-end mixer. 
	NOTE: For MCF7GFP cells, 0.5 mg/ml hIgG or 0.5 mg/ml HLA-ABC antibodies are recommended. For RAW macrophages, 1 mg/ml mCD11b are recommended at half the volume to account for the dilution differences. For Human Umbilical Vein Endothelial Cells (HUVECs), 0.5 mg/ml hCD31 is recommended.</li>
	<li>Wash the functionalized glass surface with HBSS. For this experiment, an 8 well plate is advised.
	<ol>
		<li>Add 150 &#181;l HBSS to each well for 8 well plates. Rinse twice more using HBSS. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface. Keep cold at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Add cell solutions to the wells and wait 45 min for the cells to incubate on ice on the shaker. Make the solution of biotin in sterile HBSS by using calculated volumes as described in the Supplement.</li>
	<li>Remove the cell solution from the glass wells by using HBSS.
	<ol>
		<li>Gently pipette 150 &#181;l HBSS into each well. Then pipette out the HBSS. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface.</li>
		<li>Repeat two more times. After washing, add HBSS to the wells to keep the cells wet.</li>
	</ol>
	</li>
	<li>Add 150 &#181;l of 20 mM biotin solution to each respective release well, and then wait 20 min to allow for reaction.</li>
	<li>Recollect non-specifically bound cells by washing with HBSS as stated above, and then if using fluorescently labeled cells, proceed to fluorescence image the cells. Also image live cells in a flask (as a control, to compare with the cells in the well). If using green fluorescent protein (GFP) transfected cells, the excitation will be 470 nm, and the emission will be 515 nm.</li>
</ol>
5. Antibody Optimization: Antibody Titration
<ol>
	<li>Begin by lifting the cells from the T75 or T175 flask as explained in step 4.1- 4.3. 
	NOTE: These volumes are calibrated for 24 well plates, but can be changed to suit any glass functionalized surfaces through heuristic testing. A representative antibody titration is shown in Figure 1.</li>
	<li>Count cells using the hemacytometer, and then spin down the cell solution in a conical tube at 500 x g for 5 min at 4 &#176;C. Reconstitute cells to 1 million cells per ml, using the calculations described in the Supplement.</li>
	<li>Split the 1 million cells per ml solution into six different solutions of 500 &#181;l in different centrifuge tubes for the antibody (Ab) solutions.
	<ol>
		<li>Dilute the antibody stock solution to 10 &#181;g/ml, 1 &#181;g/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml. Create the controls by using 100 &#181;l of Stain Buffer (PBS + 1% sodium azide + 1% BSA), and 500 &#181;l of cells for the control with no Ab in it. For the blank control (No Ab and no cells), use a solution of 300 &#181;l PBS. 
		NOTE: CAUTION: Sodium azide is extremely toxic, explosive, and extreme care must be taken when using it. Please consult the material safety data sheet (MSDS) and use the appropriate safety equipment.</li>
	</ol>
	</li>
	<li>Combine and incubate the Ab solutions with the cell solutions in the end-over-end mixer at 4 &#176;C for 30 min. Rinse the functionalized 24 well plates with HBSS three times. Rinse the surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface.</li>
	<li>Aliquot 125 &#181;l of each sample solution into the appropriate APTES, DSB, and SAv functionalized well. Incubate at 4 &#176;C or on ice for 45 min on the glass surface. Rinse the surface with HBSS three times to remove non-specifically attached cells. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface. Do NOT rinse the bottom row, as those are controls.</li>
	<li>Add 150 &#181;l of HBSS to each washed well, put the 24 well plates on ice, and then use a plate reader to measure fluorescence of GFP cells (Excitation 485 nm / Emission 528 nm) .</li>
</ol>
6. Cell Optimization: Cell Titration
<ol>
	<li>Lift the cells as mentioned in steps 4.1 -4.3.</li>
	<li>Count cells using a hemacytometer. Spin down cell solution in conical tube at 500 x g for 5 min at 4 &#176;C. Reconstitute cells to 1 million cells per ml. 
	NOTE: More information on the use of the hemacytometer can be found in the Supplement.</li>
	<li>Pipette 1.6 ml from the cell solution for the 1.6 million cells stock and place in a conical tube. Pipette 800 &#181;l of cells from the cell solution for the 800,000 cells stock and place in a conical tube. Pipette 80 &#181;l from the cell stock for the 80,000 cells stock and place in a conical tube. Pipette 8 &#181;l from the cell stock for 8,000 cells stock and place in a conical tube. 
	NOTE: Concentrations are given per 8 well plate measurements, replicate as needed. An example of plate output is shown in Figure 2.</li>
	<li>Take the four stock solutions and spin in a centrifuge at 500 x g for 5 min at 4 &#176;C. Re-suspend all stock solutions in 400 &#181;l of HBSS.</li>
	<li>Add 1.6 &#181;l of 100 ng/ml antibody to the 1.6 million cell stock, 0.8 &#181;l to the 800,000 cell stock, and 0.5 &#181;l to all other stocks. Incubate the antibody for 30 min in the end-over-end mixer for 30 min at 4 &#176;C. Wash the functionalized glass surface with HBSS three times. 
	NOTE: The functionalized 8 well plate is recommended for microscopy quantification, while the functionalized 24 well plate is recommended for plate reader quantification. Antibody concentration for 8,000 cell stock was not reduced to allow the limiting factor of the capture surface to not be the lack of antibodies in solution, but rather the capturing properties of the surface.</li>
	<li>Apply 150 &#181;l of the sample solutions to each well. Apply the 1.6 million cell stock to the first column of wells on the left. Apply the 800,000 cell stock to the second column from the left. Apply the 80,000 cell stock to the third column from the left. Finally apply the 8,000 cell stock to the last column. Incubate for 45 min at 4 &#176;C on the shaker. 
	NOTE: The 1.6 million cell stock serves as the highest concentration to be tested, and is double the amount of cells that is used typically in cell separation experiments (600,000 cells per well) The 800,000 cell stock serves as the control for the experiment as it is the typical cellular concentration that is used in cell separation experiments (3,000,000 cells per well). The 80,000 cell stock serves as a ten-fold dilution to test for lower concentrations for cellular capture (30,000 cells per well). The 8,000 cell stock serves as a hundred fold dilution to use as a test to check the lower limit of cellular separation (3,000 cells per well).</li>
	<li>Rinse with HBSS three times. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70&#176; angle so the tip is not directly pointed into the surface. Collect all washes.</li>
	<li>Image the cells on a fluorescence microscope, if using 8 well plates, take pictures of each surface, and apply 150&#160;&#181;l biotin to each surface for one hour at 4 &#176;C on the shaker. After one hour, rinse biotin release wells with HBSS 3 times as before. Collect all washes from wells for counting with the hemocytometer and image the release wells.</li>
	<li>Apply 150&#160;&#181;l of 20 mM excess biotin solution to appropriate biotin release wells for 1 hr, if using 24 well plates, and then rinse those wells 3 times with HBSS. Quantitate 24 well plates using a plate reader. Use a hemacytometer to count cells from all collected well washes.</li>
</ol>
7. Image Analysis
Note: The FIJI software package (http://fiji.sc/Fiji) is recommended for image analysis. Initially, the images were converted into grayscale image, and then the brightness/contrast was altered to bring out the cells.
<ol>
	<li>Load up the image, and convert to grayscale by clicking the image tab then scrolling down to &#34;Type&#34; and then clicking &#34;8 bit&#34;.</li>
	<li>Increase the contrast of the image by clicking the image tab and then scrolling to &#34;Adjust&#34;. Click on &#34;Brightness and Contrast&#34;, and then use the scroll bars to make the cells stand out. If using fluorescence images, invert the images to make the cells more defined.</li>
	<li>Load a plugin onto ImageJ called ITCN (http://rsb.info.nih.gov/ij/plugins/itcn.html) to analyze the images.</li>
	<li>Open ITCN. When a dialog box appears that prompts the user with several parameters to estimate the size of the cell, set the minimum width of the cell of interest first. Click the &#34;Detect Dark Peaks&#34; option if the image is fluorescent and the cells are darkened.</li>
	<li>Set the threshold value at 2 initially, and then hit the &#34;Count&#34; button. A newly counted version of the picture will appear with red dots delineating where cells have been counted.</li>
	<li>Adjust the threshold and width parameter to get more accurate cellular data by defining the size of a &#34;cell&#34;. Note: The number of cells counted will be in a dialog box on the right. Changing parameters will give different number of cells counted.</li>
	<li>Continue to iterate through the threshold and width parameters until a good estimate is reached for the number of cells.</li>
</ol>

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Bioeng. 104 (2), 360-370 (2009).</li><li>Perritt, D., Wong, P., Macpherson, J. L., Henrichsen, K., Symonds, G., Pond, 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> Processing Blood. , (2014).</li><li>Fukuda, S., Schmid-Sch&#246;nbein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+attenuates+the+fluid+shear+response+of+circulating+leukocytes.">Centrifugation attenuates the fluid shear response of circulating leukocytes.</a> J. Leukoc. Biol. 72 (July), 133-139 (2002).</li><li>dela Paz, N. G., Walshe, T. E., Leach, L. L., Saint-Geniez, M., D'Amore, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+shear-stress-induced+VEGF+expression+in+endothelial+cell+survival.">Role of shear-stress-induced VEGF expression in endothelial cell survival.</a> J. Cell Sci. 125 (Pt 4), 831-843 (2012).</li><li>Allard, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Cells+Circulate+in+the+Peripheral+Blood+of+All+Major+Carcinomas+but+not+in+Healthy+Subjects+or+Patients+With+Nonmalignant+Diseases+Tumor+Cells+Circulate+in+the+Peripheral+Blood+of+All+Major+Carcinomas+but+not+in+Healthy+Subjects+or+Patients+With+Nonmalignant+diseases.&amp;#34.">Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant diseases.&amp;#34.</a> Clinical Cancer Research. 10, 6897-6904 (2005).</li><li>Nagrath, 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=Isolation+of+rare+circulating+tumour+cells+in+cancer+patients+by+microchip+technology.">Isolation of rare circulating tumour cells in cancer patients by microchip technology.</a> Nature. 450 (7173), 1235-1239 (2007).</li><li>Chen, S., Weddel, J., Gupta, P., Conard, G., Parkin, J., Imoukhuede, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QFlow+Cytometer-Based+Receptoromic+Screening:+A+High-throughput+Quantification+Approach+Informing+Biomarker+Selection+and+Nanosensor.">QFlow Cytometer-Based Receptoromic Screening: A High-throughput Quantification Approach Informing Biomarker Selection and Nanosensor.</a> Submiss. , (2016).</li><li>Vasa, 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=Number+and+migratory+activity+of+circulating+endothelial+progenitor+cells+inversely+correlate+with+risk+factors+for+coronary+artery+disease.">Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.</a> Circ. Res. 89 (1), E1-E7 (2001).</li><li>Hirsch, J. D., Eslamizar, L., 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=Easily+reversible+desthiobiotin+binding+to+streptavidin,+avidin,+and+other+biotin-binding+proteins:+uses+for+protein+labeling,+detection,+and+isolation.">Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.</a> Anal. Biochem. 308 (2), 343-357 (2002).</li><li>Wilchek, M., Bayer, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Avidin-Biotin+Technology:+Literature+Survey.">Applications of Avidin-Biotin Technology: Literature Survey.</a> Methods Enzymol. 152 (1), 183-189 (1987).</li><li>Wu, 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=Immunofluorescent+labeling+of+cancer+marker+Her2+and+other+cellular+targets+with+semiconductor+quantum+dots.">Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.</a> Nat. Biotechnol. 21 (1), 41-46 (2002).</li><li>Lee-Montiel, F. T., Imoukhuede, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+quantum+dot+calibration+standards+for+quantitative+fluorescent+profiling.">Engineering quantum dot calibration standards for quantitative fluorescent profiling.</a> J. Mater. Chem. B. 1, 6434 (2013).</li><li>Hornes, E., Korsnes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oligonucleotide-linked magnetic particles and uses thereof. , (1996).</li><li>Naranbhai, V., 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=Impact+of+blood+processing+variations+on+natural+killer+cell+frequency,+activation,+chemokine+receptor+expression+and+function.">Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function.</a> J. Immunol. Methods. 366 (1-2), 28-35 (2011).</li><li>Yadav, A. R., Sriram, R., Carter, J. A., Miller, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+solution-phase+and+vapor-phase+deposition+of+aminosilanes+on+silicon+dioxide+surfaces.">Comparative study of solution-phase and vapor-phase deposition of aminosilanes on silicon dioxide surfaces.</a> Mater. Sci. Eng. C. 35 (1), 283-290 (2014).</li></ol>

The authors have nothing to disclose.

Improvements in cell isolation techniques furthers scientific studies in structure-function relationships in neuroscience18, stem cell programming in regenerative biology, and angiogenic signaling in vascular biology19. Indeed, primary cell culture20 (e.g., HUVECs) in vascular biology is primarily done through the use of cell isolation techniques. Cell isolation was also recently used for quantitative flow (qFlow) cytometry analysis of plasma membrane receptors3,14,15,19,21<...

Current bench-top cell separation approaches (e.g., fluorescence activated cell sorting1, laser capture micro-dissection2, immuno-magnetic bead separation1) can take several hours of preparation and sorting. These large time scales can affect physiological response and expression levels, resulting in analyses that are not representative of the physiological response3. Systems are needed that can rapidly and efficiently isolate specific cell types without disrupting cell-surface receptor-levels in order to improve cell isolation and enrichment for biomedical applications. Therefore, the rationale for our approach is to develop a gentle approach for cell isolation.
The &#34;lab on a chip&#34; concept offers the promise of orders of magnitude quicker (hours-to-minutes) cell isolation, and most frequently involves capturing cells onto a surface and releasing cells or intracellular contents through physical4,5 or chemical methods6. Although these approaches offer a few advantages such as identifying protein7,8 expression, identifying RNA expression9-11, or even providing cells for in vitro culture12,13, many of these techniques cannot be translated to diagnostics such as cell receptor profiling due to their non-physiological environments. Enzymatic lifting agents such as collagenases can also affect these receptor quantities14,15, meaning cell receptor quantification techniques that use these lifting agents will not generate accurate physiological data. Cellular lysis prevents differentiation between the native surface receptors, and those which were previously internalized16. This protocol describes a fast and gentle approach for cell isolation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Aminopropyl) triethoxysilane (APTES)</td>
			<td>Acros Organics</td>
			<td>919-30-2</td>
			<td>Used to make 2% APTES solution</td>
		</tr>
		<tr>
			<td>Plasma Cleaner Pico</td>
			<td>Diener</td>
			<td>Model 1</td>
			<td>Cleans surfaces and allows for bonding of PDMS to glass</td>
		</tr>
		<tr>
			<td>d-Desthiobiotin (DSB)</td>
			<td>Sigma</td>
			<td>D20655</td>
			<td>Used as the releasing mechanism in the cellular capture surface.</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>British Drug Houses (BDH)</td>
			<td>BDH1115-1LP</td>
			<td>Dissolves the DSB into solution</td>
		</tr>
		<tr>
			<td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Thermo-Scientific</td>
			<td>5g: 22980 
			25g: 22981</td>
			<td>Activates Carboxylic Acids and allows binding of proteins to glass surface.</td>
		</tr>
		<tr>
			<td>uncoated 8-well culture slide</td>
			<td>BD Falcon</td>
			<td>Case of 24: 354118 
			Case of 96: 354108</td>
			<td>Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments</td>
		</tr>
		<tr>
			<td>Glass bottom 24-well plates</td>
			<td>MatTek</td>
			<td>P24G-0-13-F</td>
			<td>Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments</td>
		</tr>
		<tr>
			<td>Mercaptoethanol</td>
			<td>Science Lab</td>
			<td>60-24-2</td>
			<td>Used to quench reaction between EDC and DSB</td>
		</tr>
		<tr>
			<td>4-Morpholinoethanesulfonic acid hydrate 
			(MES Hydrate 99%)</td>
			<td>Fisher Scientific</td>
			<td>AC172590250</td>
			<td>Used to make 0.1 M MES Buffer for use in EDC reaction</td>
		</tr>
		<tr>
			<td>Precision Oven</td>
			<td>Thermo Scientific</td>
			<td>11-475-153</td>
			<td>Used in curing of PDMS and APTES layer.</td>
		</tr>
		<tr>
			<td>Titramax 1000 Shaker</td>
			<td>Heidolph</td>
			<td>13-889-420</td>
			<td>Used to ensure even distribution of APTES on surfaces.</td>
		</tr>
		<tr>
			<td>1X Streptavidin 5mg 
			[e7105-5mg]</td>
			<td>Proteo Chem</td>
			<td>9013-20-1</td>
			<td>Biotin-binding protein 
			May cause irritation</td>
		</tr>
		<tr>
			<td>5 cm Glass Dish</td>
			<td>Fisher Scientific</td>
			<td>08748A</td>
			<td>Used in HUVEC studies as well as future profiling studies.</td>
		</tr>
		<tr>
			<td>14 cm Petri Dish with Cover</td>
			<td>Sigma-Aldrich</td>
			<td>Z717231</td>
			<td>Used to hold samples being functionalized and transport them.</td>
		</tr>
		<tr>
			<td>MCF7-GFP cells</td>
			<td>Cell Biolabs</td>
			<td>AKR211</td>
			<td>Stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>RAW264.7 
			mouse macrophages</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td>Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Life Technologies</td>
			<td>12605036</td>
			<td>Stored in 100mL at room temperature</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium</td>
			<td>Cell Media Facility at School of Chemical Sciences at UIUC</td>
			<td>50003PC</td>
			<td>Supplier: Corning</td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Cell Media Facility at School of Chemical Sciences at UIUC</td>
			<td>25-025-CI</td>
			<td>Already added into DMEM by facility. 
			Supplier: Corning</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>03-600-511</td>
			<td>Stored in 500mL at &#60; -10&#8304;C</td>
		</tr>
		<tr>
			<td>1% Penicillin&#8211;Streptomycin</td>
			<td>Life Sciences Storeroom at UIUC</td>
			<td>17602e</td>
			<td>Supplier: VWR 
			Stored in 100 ml at 4&#8304;C</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>12-565-58</td>
			<td>Small 23cm 50 pack</td>
		</tr>
		<tr>
			<td>Cell Dissociation Solution</td>
			<td>Corning</td>
			<td>MT-25-056CI</td>
			<td>Used to lift cells non-enzymatically for the use in cell experiments</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td>Used to count cells for quantification of cell solutions and capture and release effectivity.</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Amresco</td>
			<td>58-85-5</td>
			<td>Used to release cells from surface.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Created from Recipe</td>
			<td>N/A</td>
			<td>Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. (Adapted from Cold Spring Harbor Protocols): In 500 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 500 mL, filter, and then refrigerate.</td>
		</tr>
		<tr>
			<td>HLA-ABC Antibody</td>
			<td>BioLegend</td>
			<td>311402</td>
			<td>Antibody used to capture MCF7gfp cells</td>
		</tr>
		<tr>
			<td>hIgG Antibody</td>
			<td>BioLegend</td>
			<td>HP6017</td>
			<td>Antibody used to capture MCF7gfp cells</td>
		</tr>
		<tr>
			<td>MCF7 GFP cells</td>
			<td>Cell Biolabs</td>
			<td>AKR-211</td>
			<td>Luminal Breast Cancer line that has been transfected with green fluorescent protein.</td>
		</tr>
		<tr>
			<td>Assorted Conicals</td>
			<td>Thermo-Scientific</td>
			<td>15mL: 12-565-268</td>
			<td>50/15 mL plastic conicals for storing solutions and aliquots.</td>
		</tr>
		<tr>
			<td>Mini-Tube Rotators (End over End Mixer)</td>
			<td>Fisher Scientific</td>
			<td>05-450-127</td>
			<td>Used to incubate antibody and mix other cellular solutions in order to mix</td>
		</tr>
		<tr>
			<td>Axiovert 200M (Fluorescence Microscope)</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Zeiss Axiovert 200 M inverted florescence microscope.</td>
		</tr>
		<tr>
			<td>Zeba Desalting columns</td>
			<td>Thermo-Scientific</td>
			<td>PI-87770</td>
			<td>Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf</td>
		</tr>
		<tr>
			<td>EZ Link Sulfo NHS Low Weight Biotinylation Kit</td>
			<td>Thermo- Scientific</td>
			<td></td>
			<td>Used to biotinylate antibodies to allow them to integrate with the capture surface</td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>BioTek</td>
			<td>Synergy HTX Multimode Reader</td>
			<td>Used to quantitatively measure fluorescent intensity in the titration experiments.</td>
		</tr>
	</tbody>
</table>

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.

Using this protocol we show cell capture (Figure 3A) and cell release (Figure 3C) of MCF7GFP cells as well as live cell controls (Figure 4). We quantified the cell capture as 60% and 80% were released (Figure 3C). When we extended this approach to a mixture of RAW 264.7 macrophages and MCF7GFP cells, 50% of RAW macrophages were captured (Fig. 3D) and 80% of RAW macrophages were release with 20 mM biotin (...

Watch this Scientific Journal Video about A Method of Targeted Cell Isolation via Glass Surface Functionalization at JoVE.com

A Method of Targeted Cell Isolation via Glass Surface Functionalization

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 ...

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Cancer Research

9.3K Views.  University of Illinois at Urbana-Champaign. 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. 

Video: A Method of Targeted Cell Isolation via Glass Surface Functionalization

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...