Name: functionalization of atomic force microscope cantilevers with single-t cells or single-particle for immunological single-cell force spectroscopy
Uploaded: 2019-07-10T13:30:00
Description: Watch this Scientific Journal Video about Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy at JoVE.com

This work is supported by the National Natural Science Foundation of China General Program (31370878), State Key Program (31630023) and Innovative Research Group Program (81621002).

The mouse experiment protocol follows the animal care guidelines of Tsinghua University
1. Cantilever functionalization with single T cells
<ol>
	<li>Mouse spleen cells preparation
	<ol>
		<li>Sacrifice the mouse (8-16 weeks of age (either sex); e.g., C57BL/6 strain) using carbon dioxide, followed by cervical dislocation.</li>
		<li>Clean the mouse with 75% ethanol and make a midline skin incision followed by splenectomy.</li>
		<li>Homogenize the spleen in 4 mL of PBS containing 2% fetal bo...

<ol>
	<li>Benoit, M., Gabriel, D., Gerisch, G., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrete+interactions+in+cell+adhesion+measured+by+single-molecule+force+spectroscopy.">Discrete interactions in cell adhesion measured by single-molecule force spectroscopy.</a> Nature Cell Biology. 2 (6), 313-317 (2000).</li><li>Chen, 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=Strong+adhesion+by+regulatory+T+cells+induces+dendritic+cell+cytoskeletal+polarization+and+contact-dependent+lethargy.">Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.</a> Journal of Experimental Medicine. 214 (2), 327-338 (2017).</li><li>Liu, 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=Mechanotransduction-modulated+fibrotic+microniches+reveal+the+contribution+of+angiogenesis+in+liver+fibrosis.">Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis.</a> Nature Materials. 16 (12), 1252-1261 (2017).</li><li>Mu, L. 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=A+phosphatidylinositol+4,5-bisphosphate+redistribution-based+sensing+mechanism+initiates+a+phagocytosis+programing.">A phosphatidylinositol 4,5-bisphosphate redistribution-based sensing mechanism initiates a phagocytosis programing.</a> Nature Communications. 9, (2018).</li><li>Qi, C., 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=Pathology-targeted+cell+delivery+via+injectable+micro-scaffold+capsule+mediated+by+endogenous+TGase.">Pathology-targeted cell delivery via injectable micro-scaffold capsule mediated by endogenous TGase.</a> Biomaterials. 126, 1-9 (2017).</li><li>Muller, D. J., Helenius, J., Alsteens, D., Dufrene, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+probing+surfaces+of+living+cells+to+molecular+resolution.">Force probing surfaces of living cells to molecular resolution.</a> Nature Chemical Biology. 5 (6), 383-390 (2009).</li><li>Muller, D. J., Dufrene, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy:+a+nanoscopic+window+on+the+cell+surface.">Atomic force microscopy: a nanoscopic window on the cell surface.</a> Trends in Cell Biology. 21 (8), 461-469 (2011).</li><li>Radotic, 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=Atomic+force+microscopy+stiffness+tomography+on+living+Arabidopsis+thaliana+cells+reveals+the+mechanical+properties+of+surface+and+deep+cell-wall+layers+during+growth.">Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.</a> Biophysics Journal. 103 (3), 386-394 (2012).</li><li>Kuznetsova, T. G., Starodubtseva, M. N., Yegorenkov, N. I., Chizhik, S. A., Zhdanov, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+probing+of+cell+elasticity.">Atomic force microscopy probing of cell elasticity.</a> Micron. 38 (8), 824-833 (2007).</li><li>Scheuring, S., Dufrene, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy:+probing+the+spatial+organization,+interactions+and+elasticity+of+microbial+cell+envelopes+at+molecular+resolution.">Atomic force microscopy: probing the spatial organization, interactions and elasticity of microbial cell envelopes at molecular resolution.</a> Molecular Microbiology. 75 (6), 1327-1336 (2010).</li><li>Berdyyeva, T. K., Woodworth, C. D., Sokolov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+epithelial+cells+increase+their+rigidity+with+ageing+in+vitro:+direct+measurements.">Human epithelial cells increase their rigidity with ageing in vitro: direct measurements.</a> Physics in Medicine and Biology. 50 (1), 81-92 (2005).</li><li>Sokolov, I., Dokukin, M. E., Guz, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+quantitative+measurements+of+the+elastic+modulus+of+biological+cells+in+AFM+indentation+experiments.">Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments.</a> Methods. 60 (2), 202-213 (2013).</li><li>Bozna, B. 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=Binding+strength+and+dynamics+of+invariant+natural+killer+cell+T+cell+receptor/CD1d-glycosphingolipid+interaction+on+living+cells+by+single+molecule+force+spectroscopy.">Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.</a> Journal of Biological Chemistry. 286 (18), 15973-15979 (2011).</li><li>Flach, T. 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=Alum+interaction+with+dendritic+cell+membrane+lipids+is+essential+for+its+adjuvanticity.">Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.</a> Nature Medicine. 17 (4), 479-487 (2011).</li><li>Ng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-independent,+direct+membrane+binding+leads+to+cell-surface+lipid+sorting+and+Syk+kinase+activation+in+dendritic+cells.">Receptor-independent, direct membrane binding leads to cell-surface lipid sorting and Syk kinase activation in dendritic cells.</a> Immunity. 29 (5), 807-818 (2008).</li><li>Helenius, J., Heisenberg, C. P., Gaub, H. E., Muller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+force+spectroscopy.">Single-cell force spectroscopy.</a> Journal of Cell Science. 121 (11), 1785-1791 (2008).</li><li>Litvinov, R. I., Shuman, H., Bennett, J. S., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+strength+and+activation+state+of+single+fibrinogen-integrin+pairs+on+living+cells.">Binding strength and activation state of single fibrinogen-integrin pairs on living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (11), 7426-7431 (2002).</li><li>Evans, E., Ritchie, K., Merkel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+Force+Technique+to+Probe+Molecular+Adhesion+and+Structural+Linkages+at+Biological+Interfaces.">Sensitive Force Technique to Probe Molecular Adhesion and Structural Linkages at Biological Interfaces.</a> Biophysics Journal. 68 (6), 2580-2587 (1995).</li><li>Lamprecht, C., Hinterdorfer, P., Ebner, 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+biosensing+atomic+force+microscopy+in+monitoring+drug+and+nanoparticle+delivery.">Applications of biosensing atomic force microscopy in monitoring drug and nanoparticle delivery.</a> Expert Opinion on Drug Delivery. 11 (8), 1237-1253 (2014).</li><li>Charras, G. T., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+mechanotransduction+and+its+modulation+analyzed+by+atomic+force+microscope+indentation.">Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation.</a> Biophysics Journal. 82 (6), 2970-2981 (2002).</li><li>Sun, M. Z., 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=Multiple+membrane+tethers+probed+by+atomic+force+microscopy.">Multiple membrane tethers probed by atomic force microscopy.</a> Biophysics Journal. 89 (6), 4320-4329 (2005).</li><li>Yan, J. C., Liu, B., Shi, Y., Qi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+II+MHC-independent+suppressive+adhesion+of+dendritic+cells+by+regulatory+T+cells+in+vivo.">Class II MHC-independent suppressive adhesion of dendritic cells by regulatory T cells in vivo.</a> Journal of Experimental Medicine. 214 (2), 319-326 (2017).</li><li>Hao, J. 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=Phospholipase+C-mediated+hydrolysis+of+PIP2+releases+ERM+proteins+from+lymphocyte+membrane.">Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane.</a> Journal of Cell Biology. 184 (3), 451-462 (2009).</li><li>Rodriguez, R. 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=Lymphocyte-T+Adhesion+to+Fibronectin+(Fn)+-+a+Possible+Mechanism+for+T-Cell+Accumulation+in+the+Rheumatoid+Joint.">Lymphocyte-T Adhesion to Fibronectin (Fn) - a Possible Mechanism for T-Cell Accumulation in the Rheumatoid Joint.</a> Clinical and Experimental Immunology. 89 (3), 439-445 (1992).</li><li>Kimura, A., Ersson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Lymphocytes-T+by+Lectins+and+Carbohydrate-Oxidizing+Reagents+Viewed+as+an+Immunological+Recognition+of+Cell-Surface+Modifications+Seen+in+the+Context+of+Self+Major+Histocompatibility+Complex+Antigens.">Activation of Lymphocytes-T by Lectins and Carbohydrate-Oxidizing Reagents Viewed as an Immunological Recognition of Cell-Surface Modifications Seen in the Context of Self Major Histocompatibility Complex Antigens.</a> European Journal of Immunology. 11 (6), 475-483 (1981).</li><li>Miller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stimulation+of+Human+Lymphocyte-B+and+Lymphocyte-T+by+Various+Lectins.">The Stimulation of Human Lymphocyte-B and Lymphocyte-T by Various Lectins.</a> Immunobiology. 165 (2), 132-146 (1983).</li><li>Vitte, J., Pierres, A., Benoliel, A. M., Bongrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+quantification+of+the+modulation+of+interaction+between+cell-+or+surface-bound+LFA-1+and+ICAM-1.">Direct quantification of the modulation of interaction between cell- or surface-bound LFA-1 and ICAM-1.</a> Journal of Leukocyte Biology. 76 (3), 594-602 (2004).</li><li>Beaussart, A., 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=Quantifying+the+forces+guiding+microbial+cell+adhesion+using+single-cell+force+spectroscopy.">Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy.</a> Nature Protocols. 9 (5), 1049-1055 (2014).</li><li>Shu, F., 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=Cholesterol+Crystal-Mediated+Inflammation+Is+Driven+by+Plasma+Membrane+Destabilization.">Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.</a> Frontiers in Immunology. 9, (2018).</li><li>Hosseini, B. 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=Immune+synapse+formation+determines+interaction+forces+between+T+cells+and+antigen-presenting+cells+measured+by+atomic+force+microscopy.">Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (42), 17852-17857 (2009).</li></ol>

AFM-based single-cell force spectroscopy has evolved to be a powerful tool to address the biophysical properties of living cells. For those applications, the cantilever needs to be functionalized properly in order to probe specific interactions or properties on the cells of interest. Here, the methods for coupling single T cell and single micron-sized bead to the tip-less cantilever are described respectively. To attach a single T cell to the cantilever, a biocompatible glue was chosen as cell adhesive. It is a specially...

Atomic force microscopy (AFM), a versatile tool, has found many applications in cell biology research1,2,3,4,5. Apart from its high-resolution imaging capability, the native force-probing feature allows biophysical properties of living cells to be investigated directly in situ at the single-cell level6,7. These include the rigidities of subcellular structures or even whole cells8,9,10,11,12, specific ligand/receptor binding strengths at the single-molecule level on the cell surface13, and adhesion forces between single-pairs of solid particles and cells or between two cells1,2,14,15. The latter two are often categorized as single-cell force spectroscopy (SCFS)16. Owing to the readily available cantilevers with various spring constant, the force range accessible to AFM is rather broad from a few piconewtons (pN) to micronewtons (&#181;N), which adequately covers the entire range of cellular events involving forces from a few tens of pN, such as receptor-based single-molecule binding, to nN, such as phagocytic cellular events15. This large dynamic force range makes AFM advantageous over other force-probing techniques such as optical/magnetic tweezers and a biomembrane force probe, as they are more suitable for weak-force measurements, with force typically less than 200 pN17,18. In addition, AFM can function as a high-precision manipulator to deliver various stimuli onto single cells in a spatiotemporally defined manner4,19. This is desirable for the real-time single-cell activation studies. Combined with live-cell fluorescence imaging, the subsequent cellular response to the specific stimulus can be monitored concurrently, thus making AFM-based SCFS exceedingly robust as optical imaging providing a practical tool to probe cellular signaling. For instance, AFM was used to determine the strains required to elicit calcium transients in osteoblasts20. In this work, calcium transients were tracked fluorescently through calcium ratiometric imaging after the application of localized forces on cultured osteoblasts with an AFM tip. Recently, AFM was employed to stretching collagen fibrils on which hepatic stellate cells (HSC) were grown and this mechano-transduced HSC activation was real-time monitored by a fluorescent Src biosensor, whose phosphorylation as represented by the fluorescence intensity of the biosensor is correlated with HSC activation3.
In AFM-based SCFS experiments, proper functionalization of AFM cantilevers is a key step toward successful measurements. Since our research interest focuses on immune cells activation, we routinely functionalize cantilevers with particulate matters such as single solid particles that can trigger phagocytosis and/or strong immune responses4,14,15 and single T cells that can form an immune synapse with antigen presenting cells, such as activated dendritic cells (DC)2. Single solid particles are normally coupled to a cantilever via an epoxy glue in the air environment, whereas single T cells, due to their non-adhesive nature, are functionalized to a cantilever via a biocompatible glue in solution. Here, we describe the methods to perform these two types of cantilever modification and give two associated applications as well. The first application is to probe T cell/DC interactions with AFM-SCFS to understand the suppressive mechanism of regulatory T cells from the cell mechanics point of view. The second one involves combining AFM with live-cell fluorescence imaging to monitor the cellular response of macrophage to a solid particle in real-time to reveal the molecular mechanism of receptor-independent phosphatidylinositol 4,5-bisphosphate (PIP2)-Moesin mediated phagocytosis. The aim of this protocol is to provide a reference framework for interested researchers to design and implement their own experimental settings with AFM-based single-cell analysis for immunological research.

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;l pipette tip</td>
			<td>Thermo Fisher</td>
			<td>104-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm diameter culture dish</td>
			<td>NALGENE nunc</td>
			<td>150462</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>JET</td>
			<td>TCP011006</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM Cantilever</td>
			<td>NanoWorld</td>
			<td>Arrow-TL1-50</td>
			<td>tipless cantilever</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>7604</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocompatible glue</td>
			<td>BD Cell-Tak</td>
			<td>354240</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation Cocktail</td>
			<td>STEMCELL</td>
			<td>19852C.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DC2.4 cell line</td>
			<td>A gift from K. Rock (University of Massachusetts Medical School, Worcester, MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-coated magnetic particles</td>
			<td>STEMCELL</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>GENEray</td>
			<td>Generay-E1101-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>ERGO</td>
			<td>7100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>twbio</td>
			<td>00019</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Ex Cell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocker</td>
			<td>STEMCELL</td>
			<td>18731</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>local vender (Hai Men Lian Sheng)</td>
			<td>HX-E37</td>
			<td>24mm diameter, 0.17mm thinckness</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>JinTong department of laboratory and equipment management, Haimen&#160;</td>
			<td>N/A</td>
			<td>customized</td>
		</tr>
		<tr>
			<td>H2O2 (30%)</td>
			<td>Sino pharm</td>
			<td>10011218</td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sino pharm</td>
			<td>80120892</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>51558</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>STEMCELL</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh nylon strainer</td>
			<td>BD Falcon</td>
			<td>REF 352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Moesin-EGFP</td>
			<td>N/A</td>
			<td></td>
			<td>cloned in laboratory</td>
		</tr>
		<tr>
			<td>Mouse CD25 Treg cell positive isolation kit</td>
			<td>STEMCELL</td>
			<td>18782</td>
			<td>Component: FcR Blocker,Regulatory T cell Positive Selection Cocktail, PE Selection Cocktail, Dextran RapidSpheres,</td>
		</tr>
		<tr>
			<td>Mouse CD4+ Tcell isolation kit</td>
			<td>STEMCELL</td>
			<td>19852</td>
			<td>Component:CD4+T cell isolation Cocktail, Streptavidin RapidSpheres, Rat Serum</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Lanyi chemical products co., LTD&#65292; Beijing</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1022-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PE selection cocktail</td>
			<td>STEMCELL</td>
			<td>18151</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>PLC&#948;-PH-mCherry</td>
			<td>Addgene</td>
			<td>36075</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres 6.0&#956;m</td>
			<td>Polysciences</td>
			<td>07312-5</td>
			<td></td>
		</tr>
		<tr>
			<td>polystyrene round bottom tube</td>
			<td>BD Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>STEMCELL</td>
			<td>13551</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7</td>
			<td>&#160;ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-2</td>
			<td>Peprotech</td>
			<td>Peprotech, 200-02-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Regulatory T cell positive selection cocktail</td>
			<td>STEMCELL</td>
			<td>18782C</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-coated magnetic particles</td>
			<td>STEMCELL</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection kit</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% EDTA</td>
			<td>Life</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>JD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x objective NA 0.8</td>
			<td>Zeiss</td>
			<td>420650-9901</td>
			<td>Plan-Apochromat</td>
		</tr>
		<tr>
			<td>Atomic force microscope</td>
			<td>JPK</td>
			<td>cellHesion200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman coulter</td>
			<td>Allegra X-12R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging</td>
			<td>home-made objective-type total internal reflection fluorescence microscop based on a Zeiss microscope stand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>HERACELL 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Zeiss</td>
			<td>Observer A1 manual</td>
			<td></td>
		</tr>
	</tbody>
</table>

functionalization of atomic force microscope cantilevers with single-t cells or single-particle for immunological single-cell force spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

Figure 4A shows typical force-distance curves from the binding interaction between single-T cell and single-DC in one approach-retract cycle. The light red curve is the extension curve and the dark red one is the retraction curve. Since the extension curve is typically used for indentation or rigidity-analysis, here only the retraction curve is concerned for cell adhesion. The minimum value (the green circle) in the curve gives a measure of the maximum adhesi...

Watch this Scientific Journal Video about Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy at JoVE.com

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Research

JoVE Journal

Immunology and Infection

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...