Name: imaging molecular adhesion in cell rolling by adhesion footprint assay
Uploaded: 2021-09-27T15:00:00
Description: Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

Dette arbejde blev støttet af Canada Foundation of Innovation (CFI 35492), Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2017-04407), New Frontiers in Research Fund (NFRFE-2018-00969), Michael Smith Foundation for Health Research (SCH-2020-0559) og University of British Columbia Eminence Fund.

1. Oligonucleotid mærkning og hybridisering
<ol><li>Reduktion af protein G disulfidbindinger<ol>
<li>10 mg protein G (ProtG) opløses i 1 mL ultrapurt vand. BEMÆRK: Protein G her er modificeret med en enkelt cystein rest på C-endestation og en N-terminus poly-histidin tag.</li>
		<li>Bufferudveksling ≥20 μL protG (10 mg/mL) i 1x PBS (pH 7.2) med en P6-kolonne.</li>
		<li>Mål proteinkoncentrationen efter bufferudveksling. BEMÆRK: Typisk koncentration på 7-8 mg/mL.</li>
		<li>Der fremstill...

<ol>
	<li>McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+cell+adhesion.">Rolling cell adhesion.</a> Annual Review of Cell and Developmental Biology. 26 (1), 363-396 (2010).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+The+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: The leukocyte adhesion cascade updated.</a> Nature Reviews Immunology. 7 (9), 678-689 (2007).</li><li>Zarbock, A., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesion+and+activation+under+flow.">Neutrophil adhesion and activation under flow.</a> Microcirculation. 16 (1), 31-42 (2009).</li><li>Etzioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+molecules+-+Their+role+in+health+and+disease.">Adhesion molecules - Their role in health and disease.</a> Pediatric Research. 39 (2), 191-198 (1996).</li><li>van de Vijver, E., vanden Berg, T. K., Kuijpers, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+adhesion+deficiencies.">Leukocyte adhesion deficiencies.</a> Hematology/Oncology Clinics of North America. 27 (1), 101-116 (2013).</li><li>Hanna, S., Etzioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+adhesion+deficiencies.">Leukocyte adhesion deficiencies.</a> Annals of the New York Academy of Sciences. 1250 (1), 50-55 (2012).</li><li>Geng, Y., Marshall, J. R., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomechanics+of+the+metastatic+cascade:+Tumor+cell-endothelial+cell+interactions+in+the+circulation.">Glycomechanics of the metastatic cascade: Tumor cell-endothelial cell interactions in the circulation.</a> Annals of Biomedical Engineering. 40 (4), 790-805 (2012).</li><li>Yasmin-Karim, S., King, M. R., Messing, E. M., Lee, 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=E-selectin+ligand-1+controls+circulating+prostate+cancer+cell+rolling/adhesion+and+metastasis.">E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis.</a> Oncotarget. 5 (23), 12097-12110 (2014).</li><li>Marshall, B. T., Long, M., Piper, J. W., Yago, T., McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+catch+bonds+involving+cell-adhesion+molecules.">Direct observation of catch bonds involving cell-adhesion molecules.</a> Nature. 423 (6936), 190-193 (2003).</li><li>Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+dynamics.">Adhesive dynamics.</a> Journal of Biomechanical Engineering. 136 (2), 021006 (2014).</li><li>Yasunaga, A. B., Murad, Y., Kapras, V., Menard, F., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+interpretation+of+cell+rolling+velocity+distribution.">Quantitative interpretation of cell rolling velocity distribution.</a> Biophysical Journal. 120 (12), 2511-2520 (2021).</li><li>Li, I. T. S., Ha, T., Chemla, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cell+surface+adhesion+by+rotation+tracking+and+adhesion+footprinting.">Mapping cell surface adhesion by rotation tracking and adhesion footprinting.</a> Scientific Reports. 7 (1), 44502 (2017).</li><li>Yasunaga, A. B., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+fast+molecular+adhesion+by+fluorescence+footprinting.">Quantification of fast molecular adhesion by fluorescence footprinting.</a> Science Advances. 7 (34), (2021).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nature Methods. 8 (12), 1027-1040 (2011).</li><li>Zhu, M., Lerum, M. Z., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+prepare+reproducible,+homogeneous,+and+hydrolytically+stable+aminosilane-derived+layers+on+silica.">How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica.</a> Langmuir. 28 (1), 416-423 (2012).</li><li>Chandradoss, S. D., Haagsma, A. C., Lee, Y. K., Hwang, J. H., Nam, J. M., Joo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments: JoVE. (86), e50549 (2014).</li><li>Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F., Jungmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+with+DNA-PAINT.">Super-resolution microscopy with DNA-PAINT.</a> Nature Protocols. 12 (6), 1198-1228 (2017).</li><li>Chalfoun, 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=MIST:+Accurate+and+scalable+microscopy+image+stitching+tool+with+stage+modeling+and+error+minimization.">MIST: Accurate and scalable microscopy image stitching tool with stage modeling and error minimization.</a> Scientific Reports. 7 (1), 4988 (2017).</li><li>Wang, 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=Constructing+modular+and+universal+single+molecule+tension+sensor+using+protein+G+to+study+mechano-sensitive+receptors.">Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors.</a> Scientific Reports. 6, 1-10 (2016).</li><li>Crockett-Torabi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectins+and+mechanisms+of+signal+transduction.">Selectins and mechanisms of signal transduction.</a> Journal of Leukocyte Biology. 63 (1), 1-14 (1998).</li><li>Ye, 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=The+P-selectin+and+PSGL-1+axis+accelerates+atherosclerosis+via+activation+of+dendritic+cells+by+the+TLR4+signaling+pathway.">The P-selectin and PSGL-1 axis accelerates atherosclerosis via activation of dendritic cells by the TLR4 signaling pathway.</a> Cell Death and Disease. 10 (7), 507 (2019).</li><li>Saha, K., Bender, F., Gizeli, E. <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+IgG+binding+to+proteins+G+and+A:+Nonequilibrium+kinetic+and+binding+constant+determination+with+the+acoustic+waveguide+device.">Comparative study of IgG binding to proteins G and A: Nonequilibrium kinetic and binding constant determination with the acoustic waveguide device.</a> Analytical Chemistry. 75 (4), 835-842 (2003).</li><li>Murad, Y., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+molecular+forces+with+serially+connected+force+sensors.">Quantifying molecular forces with serially connected force sensors.</a> Biophysical Journal. 116 (7), 1282-1291 (2019).</li><li>Yasunaga, A. B., Murad, Y., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+molecular+tension-classifications,+interpretations+and+limitations+of+force+sensors.">Quantifying molecular tension-classifications, interpretations and limitations of force sensors.</a> Physical Biology. 17 (1), 011001 (2020).</li></ol>

Vedhæftning fodaftryk assay muliggør visualisering af molekylær vedhæftning begivenheder mellem PSGL-1 og P-selectin under celle rullende vedhæftning. Denne proces er initieret af P-selectin-medieret optagelse efterfulgt af rullende under fluidisk forskydning stress. Potentielle problemer i løbet af eksperimentet involverer normalt dårlig celle rullende eller manglende fluorescerende spor, selv når cellerne ruller godt. Disse problemer skyldes ofte kvalitetskontrol ved de kritiske trin i protokollen, som angivet ...

Den rullende vedhæftning kaskade beskriver, hvordan cirkulerende celler binde til og rulle langs blodkarvæggen1. Passiv rullende er primært medieret af selectins, en stor klasse af cellulære vedhæftning molekyler (CAMs)1. Under forskydningen af blod danner leukocytter, der udtrykker P-selectin glycoprotein ligand-1 (PSGL-1), meget forbigående bindinger med P-selectin, som kan udtrykkes på overfladen af betændte endotelceller. Denne proces er afgørende for leukocytter at migrere til et sted for inflammation2. Derudover er PSGL-1 også en mechanosensitiv receptor, der er i stand til at udløse den efterfølgende faste vedhæftningsfase af den rullende vedhæftningskaskade ved dets engagement med P-selectin3.
Genetiske mutationer, der påvirker CAM-funktionen, kan alvorligt påvirke immunsystemet, såsom i den sjældne sygdom af leukocyt vedhæsionsmangel (LAD), hvor funktionsfejl i vedhæftningsmolekyler, der formidler rullende, fører til alvorligt immunkompromitterede individer4,5,6. Derudover har cirkulerende tumorceller vist sig at migrere efter en lignende rullende proces, hvilket fører til metastaser7,8. Men fordi cellerullende er hurtig og dynamisk, konventionelle eksperimentelle mekanobiologi metoder er uegnede til at studere molekylære interaktioner under celle rullende. Mens enkeltcellede og enkeltmolekyle manipulationsmetoder som atomprøvesprængningsmikroskopi og optisk pincet var i stand til at studere molekylære interaktioner som P-selectins kraftafhængige interaktion med PSGL-1 på enkeltmolekyleniveau9, er de uegnede til at undersøge levende vedhæftning hændelser under cellerullende. Derudover kan interaktionen karakteriseret in vitro ikke direkte besvare spørgsmålet om molekylær vedhæhæftning in vivo. Hvilket molekylært spændingsområde er f.eks. biologisk relevant, når celler fungerer i deres oprindelige miljø? Beregningsmetoder såsom selvklæbende dynamik simulering10 eller simpel steady-state model11 har fanget visse molekylære detaljer, og hvordan de påvirker den rullende adfærd, men er meget afhængige af nøjagtigheden af modellering parametre og antagelser. Andre teknikker såsom trækkraftmikroskopi kan detektere kræfter under cellemigration, men giver ikke tilstrækkelig rumlig opløsning eller kvantitative oplysninger om molekylær spænding. Ingen af disse teknikker kan give direkte eksperimentelle observationer af den tidsmæssige dynamik, rumlige fordeling og størrelse heterogenitet af molekylære kræfter, som direkte vedrører cellefunktion og adfærd i deres oprindelige miljø.
Derfor er implementering af en molekylær kraftsensor, der er i stand til nøjagtigt at måle udvalgteinmedierede interaktioner, afgørende for at forbedre vores forståelse af rullende vedhæftning. Her beskriver vi protokollen for vedhæftning fodaftryk assay12 hvor PSGL-1 belagt perler er rullet på en overflade præsentere p-selectin funktionaliseret spænding gauge tethers (TGTs)13. Disse TGT'er er irreversible DNA-baserede kraftsensorer, der resulterer i en permanent historie med brudhændelser i form af fluorescensudlæsning. Dette opnås gennem brud på TGT (dsDNA) og derefter efterfølgende mærkning af den bristede TGT (ssDNA) med en fluorescerende mærket komplementær streng. En stor fordel ved dette system er dets kompatibilitet med både diffraktionsbeskret og superopløsningsbilleddannelse. Den fluorescerende mærkede komplementære streng kan enten være permanent bundet (>12 bp) for diffraktionsbegrænset billeddannelse eller forbigående bundet (7-9 bp) til billeddannelse i superopløsning gennem DNA PAINT. Dette er et ideelt system til at studere rullende vedhæftning som TGTs er bristet under aktiv rullende, men fluorescens udlæsning er analyseret post-rullende. De to billeddannelsesmetoder giver også brugeren mere frihed til at undersøge rullende vedhæftning. Typisk, diffraktion-begrænset billeddannelse er nyttig til udvinding molekylær brudkraft gennem fluorescens intensitet13, mens super-opløsning billeddannelse giver mulighed for kvantitativ analyse af receptortæthed. Med evnen til at undersøge disse egenskaber ved rullende vedhæftning giver denne tilgang en lovende platform for forståelse af kraftreguleringsmekanismen på den molekylære vedhæftning af rullende celler under forskydningsstrømning.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-channel drill guide</td>
			<td>Custom made</td>
			<td></td>
			<td>3D printed with ABS filament</td>
		</tr>
		<tr>
			<td>4-holes slide</td>
			<td>Custom made</td>
			<td></td>
			<td>Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>VWR</td>
			<td>BDH1101-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic spacer</td>
			<td>Custom made</td>
			<td></td>
			<td>Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder.</td>
		</tr>
		<tr>
			<td>Aluminium chip holder</td>
			<td>Custom made</td>
			<td></td>
			<td>Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread.</td>
		</tr>
		<tr>
			<td>Aminosilane</td>
			<td>AlfaAesar</td>
			<td>L14043</td>
			<td>CAS 1760-24-3</td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic solution</td>
			<td>Cytiva HyClone</td>
			<td>SV3007901</td>
			<td>Pen/Strep/Fungiezone</td>
		</tr>
		<tr>
			<td>Beads, ProtG coated polystyrene</td>
			<td>Spherotech</td>
			<td>PGP-60-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>VWR</td>
			<td>332</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer, DNA PAINT</td>
			<td></td>
			<td></td>
			<td>0.05% Tween-20, 5 mM Tris, 75 mM MgCl2, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Buffer, T50M5</td>
			<td></td>
			<td></td>
			<td>10mM Tris, 50 mM NaCl, 5 mM MgCl2</td>
		</tr>
		<tr>
			<td>Buffer, Rolling</td>
			<td></td>
			<td></td>
			<td>HBSS with 2mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 0.1% BSA</td>
		</tr>
		<tr>
			<td>Buffer, Wash</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 50 mM NaCl, 5 mM MgCl2 and 2 mM CaCl2, 0.05% Tween 20</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>VWR</td>
			<td>BDH9224</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>VWR</td>
			<td>10062-868</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated sulfuric acid</td>
			<td>VWR</td>
			<td>BDH3072-2.5LG</td>
			<td>95-98%</td>
		</tr>
		<tr>
			<td>Coverslip holding tweezers</td>
			<td>Techni-Tool</td>
			<td>758TW150</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond-coated drill bits</td>
			<td>Abrasive technology</td>
			<td>C5250510</td>
			<td>0.75 mm diamond drill</td>
		</tr>
		<tr>
			<td>DNA, amine-ssDNA (top strand)</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>CCGGGCGACGCAGGAGGG /3AmMO/</td>
		</tr>
		<tr>
			<td>DNA, biotin-ssDNA (bottom strand)</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>/5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG</td>
		</tr>
		<tr>
			<td>DNA, imager strand for DNA-PAINT</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>GAGGGAAA TT/3Cy3Sp/</td>
		</tr>
		<tr>
			<td>DNA, imager strand for permanent labelling</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>CCGGGCGACGCAGG /3Cy3Sp/</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>237</td>
			<td>3/4 inch width, permanent double-sided tape</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermofisher</td>
			<td>15575020</td>
			<td>0.5 M EDTA, pH 8.0</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Gorilla</td>
			<td>42001</td>
			<td>5 minute curing time</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Avantor</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GelGreen</td>
			<td>Biotium</td>
			<td>41005</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>VWR</td>
			<td>BDH3094-2.5LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass, Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass, Microscope slide</td>
			<td>VWR</td>
			<td>48300-026</td>
			<td>75 mm x 25 mm x 1 mm</td>
		</tr>
		<tr>
			<td>Glass, Staining jar</td>
			<td>VWR</td>
			<td>74830-150</td>
			<td>Wheaton Staining Jar (900620)</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt solution (HBSS)</td>
			<td>Lonza</td>
			<td>04-315Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity chamber slide support</td>
			<td>Custom made</td>
			<td></td>
			<td>3D printed with ABS filament</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>VWR</td>
			<td>BDH7690-1</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>Inlets/outlets</td>
			<td>Custom made</td>
			<td></td>
			<td>Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm&#160; polyethylene tubing (Tygon, I.D. 1/32&#8221; O.D. 3/32&#8221;) into each hollow setscrew</td>
		</tr>
		<tr>
			<td>Iscove Modified Dulbecco Media (IMDM)</td>
			<td>Lonza</td>
			<td>12-722F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>VWR</td>
			<td>BDH9244</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Ni-NTA beads</td>
			<td>Invitrogen</td>
			<td>10103D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mailer tubes</td>
			<td>EMS</td>
			<td>EMS71406-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Bio-Spin P-6 Gel Columns</td>
			<td>Biorad</td>
			<td>7326200</td>
			<td>In SSC Buffer</td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>Laysan Bio</td>
			<td>MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-biotin</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>VWR</td>
			<td>470302-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein, Protein G</td>
			<td>Abcam</td>
			<td>ab155724</td>
			<td>N-terminal His-Tag and C-terminal cysteine</td>
		</tr>
		<tr>
			<td>Protein, P-selectin-Fc</td>
			<td>R&#38;D System</td>
			<td>137-PS</td>
			<td>Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF</td>
		</tr>
		<tr>
			<td>Protein, Streptavidin</td>
			<td>Cedarlane</td>
			<td>CL1005-01-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump Syringe</td>
			<td>Harvard Apparatus</td>
			<td>704801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Ward&#8217;s Science</td>
			<td>470302-444</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>97061-274</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>Thermofisher</td>
			<td>22322</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>81520</td>
			<td>Syringes with PTFE luer lock, 5 mL</td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>BD</td>
			<td>305115</td>
			<td>Precision Glide 26 G, 5/8 Inch Length</td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-2G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>BDH4502-500GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing, Adaptor</td>
			<td>Tygon</td>
			<td>ABW00001</td>
			<td>Formulation 3350, I.D. 1/32&#8221;; O.D. 3/32&#8221;</td>
		</tr>
		<tr>
			<td>Tubing, Polyethylene</td>
			<td>BD Intramedic</td>
			<td>427406</td>
			<td>Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>93773</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging molecular adhesion in cell rolling by adhesion footprint assay

Rullende vedhæftning, lettet af selectin-medieret interaktioner, er en meget dynamisk, passiv motilitet i at rekruttere leukocytter til stedet for betændelse. Dette fænomen forekommer i postcapillary venules, hvor blodgennemstrømningen skubber leukocytter i en rullende bevægelse på endotelcellerne. Stabil rulning kræver en delikat balance mellem vedhæftningsbindingsdannelse og deres mekanisk drevne dissociation, så cellen kan forblive fastgjort til overfladen, mens den ruller i strømmens retning. I modsætning til andre vedhæftningsprocesser, der forekommer i relativt statiske miljøer, er rullende vedhæftning meget dynamisk, da de rullende celler bevæger sig over tusindvis af mikron med snesevis af mikron i sekundet. Derfor er konventionelle mekanobiologiske metoder såsom trækkraftmikroskopi uegnede til måling af de enkelte vedhæftning og de tilhørende molekylære kræfter på grund af den korte tidshorisont og høj følsomhed, der kræves. Her beskriver vi vores seneste implementering af vedhæftning fodaftryk assay til billede P-selectin: PSGL-1 interaktioner i rullende vedhæftning på molekylært niveau. Denne metode udnytter irreversible DNA-baserede spændingsmåler tøjre til at producere en permanent historie af molekylære vedhæftning begivenheder i form af fluorescens spor. Disse spor kan afbildes på to måder: (1) syning sammen tusindvis af diffraction-begrænsede billeder til at producere et stort synsfelt, der muliggør udvinding af vedhæftning fodaftryk af hver rullende celle over tusindvis af mikron i længden, (2) udfører DNA-PAINT at rekonstruere super-opløsning billeder af fluorescens spor inden for et lille synsfelt. I denne undersøgelse, vedhæftning fodaftryk assay blev brugt til at studere HL-60 celler rullende på forskellige forskydning understreger. Ved at gøre det var vi i stand til at forestille os den rumlige fordeling af P-selectin: PSGL-1 interaktion og få indsigt i deres molekylære kræfter gennem fluorescensintensitet. Denne metode danner således grundlag for den kvantitative undersøgelse af de forskellige celleoverfladeinteraktioner, der er involveret i rullende vedhæftning på molekylært niveau.

Protokollen ovenfor beskriver den eksperimentelle procedure for vedhæftning fodaftryk assay. Den generelle eksperimentarbejdsgang er illustreret i figur 1, fra flowkammersamlingen (figur 1A) til overfladefunktionalisering (figur 1B) og eksperiment- og billedtrin (figur 1C).
Figur 2 er et repræsentativt resultat for ProtG-ssDNA biokonjugationska...

Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Denne protokol præsenterer de eksperimentelle procedurer til at udføre vedhæftning fodaftryk assay til billede vedhæftning begivenheder under hurtig celle rullende vedhæftning.

Imaging Molekylær vedhæftning i Cell Rolling ved vedhæftning Fodaftryk Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Denne protokol hjælper med at etablere forbindelsen mellem molekylær kraft og cellerullende adfærd udover at gøre det muligt rumligt at kortlægge og kvantificere rullende vedhæftning på molekylært niveau. Denne teknik gør det muligt at studere individuelle vedhæftningshændelser under selve cellerullende, så vi kan måle den fysiologiske relevante molekylære vedhæftningskraft direkte. Overfladeforberedelse ved hjælp af den passende koncentrations- og inkubationstid i hvert trin er afgørende for at opnå overfladefunktionalisering af høj kvalitet.Kvaliteten af biokonjugationer og ordentlige forhold er også afgørende. Visuel demonstration er afgørende for at hjælpe forskere med at replikere denne protokol. Især vil trinene som flowkammersamling og efterbehandlingsbilleder drage stor fordel af en visuel demonstration.Begynd med at sprede en lille mængde epoxy på begge sider af det dobbeltsidede bånd med et barberblad. Ved hjælp af en laser skal du skære det epoxybelagte bånd for at skabe fire kanaler. Opret flow chip ved sandwiching epoxy tape mellem en fire-hullers slide og PEG coverlip.Brug en pipettespids skal du anvende blidt tryk langs kanalernes længde for at skabe en god forsegling og derefter helbrede epoxyen i mindst en time. Juster chippen, så åbningen af hver kanal placeres i midten af kortet. Placer derefter to gennemsigtige akryl spacers oven på chippen.Påfør fast tryk i midten af blokken og skrue i enderne af hver afstandstrækker. Skru indløbene ind i gevindhullerne på den anden side af beslaget, og overvåg tætningsbetingelsen gennem den gennemsigtige akrylblok. Flow 200 mikroliter af vaskebuffer ind i kammeret for at kontrollere for lækage.Hvis der dannes bobler i kanalen, skal du aggressivt skubbe yderligere 200 mikroliter vaskebuffer for at fjerne boblerne. Tilsættes 40 mikroliter BSA til strømningskammeret for at forhindre uspecifik binding og inkubation i 10 minutter i fugtighedskammeret. Efter inkubation tilsættes 40 mikroliter ved Tween 20 til flowkammeret og inkuberes igen i 10 minutter for yderligere at reducere den uspecifikke binding.Vask derefter kanalen med 200 mikroliter vaskebuffer for at fjerne alle passiveringsmidler. For kammeroverfladefunktionalisering tilsættes 40 mikroliter af Streptavidin til flowkammeret og inkuberes i 20 minutter, og vask derefter kammeret med 200 mikroliter vaskebuffer. Nu tilsættes 40 mikroliter af hybridiseret protein G-TGT til flowkammeret og inkuberes i 20 minutter.Efter vask med vaskebuffer tilsættes 40 mikroliter protein G-TGT top streng og inkuberes i 20 minutter for at fuldføre enhver uhybridiseret TGT bundstreng på overfladen og derefter vaskes med vaskebuffer. Endelig tilsættes 40 mikroliter P-selectin-Fc til flowkammeret og inkuberes i 60 minutter før vask med vaskebuffer. Fyld en fem milliliter glassprøjte med den rullende buffer og bank på siderne af sprøjten for at løsne og skubbe boblerne ud, når de flyder mod spidsen.Efter at have indsat en steril nål i et 200 millimeter stykke polyethylenrør, skal du forbinde nålen til glassprøjten. Fastgør sprøjten på sprøjtepumpen, og vip sprøjtepumpen, så stemplet er hævet for at forhindre luftbobler i at komme ind i kanalen. Sæt enden af røret ind i flowkammerets indløb.Indsæt den ene ende af en anden 200 millimeter stykke af polyethylen slange i stikkontakten og nedsænke den anden ende i et affald bæger. Tag en til to milliliter af celleaffjedringsprøven og centrifugen for at pille cellerne. Fjern mediet og forsigtigt genbruge cellerne i 500 mikroliter af den rullende buffer.Afbryd forsigtigt slangen fra indløbene og udløbet og pipetten 40 mikroliter af celleaffjedringen ind i flowkammeret. Tilslut slangen igen som beskrevet tidligere, så boblerne ikke indføres i flowkanalen. Begynd cellerullende eksperiment ved at starte sprøjtepumpen med de ønskede strømningshastigheder.Overhold cellerullende ved hjælp af et mørkt feltmikroskop med et 10X-mål. Når eksperimentet er afsluttet, skal du fjerne cellerne fra kanalen ved at tilføre den rullende buffer med 100 milliliter i timen, indtil overfladen er cellefri. Til billeddannelse af de lokale spor ved DNA-PAINT, tilføje 40 mikroliter af DNA-PAINT imager streng udarbejdet i DNA-PAINT buffer til kanalen.Udfør total intern refleksion fluorescensmikroskopi ved hjælp af de betingelser, der er nævnt i tekstmanuskriptet. Lokalisere og gengive billederne i superopløsningen. For billeddannelse af de lange spor ved permanent mærkning, tilføje den permanente imager streng og inkubere i 120 sekunder i T50M5 buffer.Vask derefter kanalen ved at tilføre 200 mikroliter vaskebuffer. Optag et billede med excitation laser off for at få baggrundskamera støj. Billede et stort område i et gitter mønster af TIRF mikroskopi.Programmer mikroskopet til at scanne over 400 med 50 billeder og opdele de rå data i individuelle TIP-filer, der hver indeholder maksimalt 10.000 billeder ved hjælp af ImageJ-programmet. Flad alle billederne ved hjælp af belysningsprofilen og brug MIST-pluginet til at sy billederne. Resultatet for proteinet G ssDNA biokonjugation karakterisering viste en næsten en er til et forhold mellem protein G til ssDNA hvor proteinet G Maleimide ssDNA og imidazole elution buffer spektre fra det ortogonale grundlag for montering til biokonjugation produkt spektrum.Native PAGE blev brugt til at bekræfte biokonjugationen, der viser de lysegrønne bånd, der falder sammen med det monomeriske protein G-bånd, der indikerer vellykket bøjning og godt udbytte. TIRF belysning profil indført fra en enkelt tilstand fiber er generelt lysere i midten af synsfeltet og lysdæmper rundt om kanterne. For at kompensere for den ujævne belysning og flade billederne til kvantitativ analyse blev belysningsprofilen bestemt af i gennemsnit tusindvis af individuelle rammer.Flade billeder blev produceret ved at trække kameraet støj fra både rå og belysning profiler og derefter normalisere ved belysning profil. Rå syninger viste klare periodiske mønstre svarende til de ikke-korrigerede billeder, mens det samme synsfelt syet fra flade billeder producerede en flad baggrund. En rampe op flow profil blev brugt til at bestemme rækken af forskydning stress resulterer i celle rullende og udbytte fluorescens spor, hvorunder en typisk enkeltcellet vedhæftning fodaftryk kunne ses.Suboptimale og optimale resultater af lysstofrør med utilstrækkelig kontrast, overdreven sportæthed, optimal sportæthed og kontrast samt diffraktion begrænset og DNA-PAINT-billeddannelse vises. inkubationstid på mere end 60 minutter sikrer overfladefunktionalisering og korrekt kammerkonstruktion forhindrer passage af bobler, der beskadiger funktionaliseret overflade. Denne procedure gælder for kvantitativ analyse af molekylær kraft, der er involveret i rullende vedhæftning og giver forskerne mulighed for at forstå den rullende adfærd af de forskellige celletyper.Denne teknik gør det muligt for forskere at udforske nye spørgsmål vedrørende hurtige vedhæftningshændelser, der fører til yderligere fremskridt af enkeltmolekyle og mekanobiologiforskning.

imaging-molecular-adhesion-in-cell-rolling-by-adhesion-footprint-assay

Research

JoVE Journal

Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Title Cell Encapsulation by Droplets

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Lev Imaging Assay for at vurdere de roller Ca<sup&gt; 2 +</sup&gt; Og sphingomyelinase i reparation af poredannende Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo 4-Dimensional sporing af hæmatopoietisk stamcelletransplantation og stamcelle i Voksen Mouse calvarie knoglemarv

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Aktivering Autophagy ved aerob træning i mus

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Analyse Cell Surface Vedhæftning Remodeling i Reaktion på Mekanisk Tension Brug magnetiske perler

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...