Nous tenons à remercier Melissa Klocke d&#39;assistance préparer la Figure 1 et Anna Ratliff pour démontrer le protocole. Ce travail a été soutenu par la Société pour l&#39;avancement de la recherche scientifique.

1. Microtubules vol à voile Assay Solutions mères Préparer à l&#39;avance de dosage glisse. <ol><li> Polymériser 0,5 mg microtubules peu marquées avec un fluorophore organique lumineuse 22. La concentration étiquette cible est de 1 fluorophore par micromètre de microtubules, soit une densité étiquetage d&#39;environ 1 fluorophore par 1.500 dimères de tubuline. Entreposer à la température ambiante, la lumière protégée avec de l&#39;aluminium, une feuille pour un maximum de deux semaines. </li><li> Purifier biotine-kinésine 21 à environ 1 uM. Conserver à -80 ° C en 5 aliquotes pour utilisation dans des expériences individuelles. </li><li> Préparer le tampon de dosage (AB) de 50 mM imidiazole, 50 mM de chlorure de potassium (KCl), 4 mM de chlorure de magnésium (MgCl 2), 2 mM d&#39;éthylène-glycol d&#39;acide tétraacétique (EGTA), pH 6,7. Filtre stérile et conserver à 4 ° C. </li><li> Dissoudre biotinylé albumine de sérum bovin (BSA-biotine) à 2 mg / ml à AB. Filtre avec filtre de 0,2 um seringue.Conserver à -80 ° C en aliquotes de 100 pi pendant de longues périodes, à 4 ° C pendant un mois. </li><li> Dissoudre la streptavidine (SA) à 10 mg / ml à AB. Filtre avec filtre de 0,2 um seringue. Conserver à -80 ° C en 20 aliquotes pendant de longues périodes, à 4 ° C pendant un maximum de deux semaines. </li><li> Dissoudre α-caséine à 5 mg / ml à AB. Filtre avec filtre de 0,2 um seringue. Conserver à -80 ° C en aliquotes de 100 pi pendant de longues périodes, à 4 ° C pendant un maximum de deux semaines. </li><li> Dissoudre dithiothréitol (DTT) dans de l&#39;eau déminéralisée à 200 mM. Conserver à -20 ° C en aliquotes de 100 pi, utiliser dans les 8 heures de décongélation. Peut-être recongelé. </li><li> Dissoudre le paclitaxel (PT) en HPLC du diméthylsulfoxyde (DMSO) à 4 mM. Boutique en 10 aliquotes à -80 ° C à long terme, de -20 ° C à court terme. Utiliser dans les 8 heures suivant la décongélation. Peut-être recongelé. </li><li> Dissoudre le glucose dans de l&#39;eau désionisée à 120 mg / ml. Conserver dans aliquotes de 100 pi à -20 ° C. Peut-être recongelé. </li><li> Disrésoudre l&#39;adénosine triphosphate (ATP) dans de l&#39;eau déminéralisée à 150 mM, pH 7,0. Boutique en 5 aliquotes à -80 ° C, utiliser dans les 8 heures de décongélation. </li><li> Préparer stocks d&#39;oxygène 100x balayage 25 par dissolution de 10.000 unités de glucose-oxydase et catalase 156.000 unités dans 600 ul du tampon de dosage total. Centrifuger brièvement dans une microcentrifugeuse à granulés solides; surnageant filtre avec filtre de 0,2 um seringue. Conserver à -80 ° C en 10 aliquotes pendant de longues périodes, à 4 ° C pendant une semaine. </li></ol> 2. Microtubules Gliding test, même Préparation de la solution Jour Préparer les solutions de 02.01 à 02.06 le jour de l&#39;expérience. Avec la pratique, les solutions de 2.2 à 2.6 peuvent être préparés pendant l&#39;écoulement des cellules lavage. Sauf indication contraire, gardez solutions mères sur la glace. <ol><li> Préparer AB, 1 ml de tampon de dosage avec 10 ul de la TNT stock (AB avec 2 mM de DTT). Conserver à température ambiante. </li><li> Préparer bio-BSA tampon, 40 &amp; mu, L d&#39;un mélange 1:1 du stock biotine-BSA et AB. Conserver à température ambiante. </li><li> Préparer un tampon BSA, mélangeant 645 ul de AB avec 5,5 ul de BSA (1 mg / ml de BSA dans AB). Conserver à température ambiante. </li><li> Préparer un tampon SA, en mélangeant 57 ul de AB avec 3 streptavidine actions ul (0,5 mg / ml de streptavidine dans AB). Conserver à température ambiante. </li><li> Préparer α-caséine tampon, mélanger 200 pi de tampon BSA, 50 ul de stock caséine et 0,8 ul de 15 uM (1 mg / ml α-caséine, 0,8 mg / ml de BSA, 50 nM d&#39;ATP dans AB) ATP. Conserver à température ambiante. </li><li> Préparer la fluorescence anti-eau de Javel tampon, mélangeant 95 pl de tampon α-caséine, 2,5 ul de stock de glucose, 1 ul mélange d&#39;oxygène 100x balayage, 1 stock paclitaxel pi, 1 pi 2-mercaptoéthanol (ßME). Ajouter ßME sous une hotte. Utilisez la fluorescence anti-eau de Javel tampon dans 1 h de préparation. ATTENTION: ßME est hautement toxique et sent mauvais. Assurez-vous d&#39;être ouvert uniquement sous la hotte. Bouteille dans le magasinl&#39;absence de lumière, la lumière dégrade ßME et sa capacité à réduire photoblanchiment. </li></ol> 3. Microtubules Gliding Assay <ol><li> Construire environ quatre voies d&#39;écoulement entre une lamelle 24 x 60 mm et 22 mm x 22 lamelle avec de la graisse à vide, extrudés à partir d&#39;une seringue à travers un embout de pipette, comme un élément d&#39;espacement. Chaque voie d&#39;écoulement doit être d&#39;environ 10 pi en volume (échelle des volumes suivants en conséquence). </li><li> Lavez 10 ul de bio-BSA tampon dans chaque couloir. Incuber 5 minutes pour permettre BSA pour revêtir les surfaces de verre. </li><li> Laver chaque voie trois fois avec 15 pi de tampon BSA pour enlever la biotine libre-BSA, tout en continuant à bloquer la surface de glissement. Utilisez un Kimwipe ou de papier filtre pour éliminer le tampon du côté opposé de chambre d&#39;écoulement, en étant sûr de ne pas laisser de bulles d&#39;air à passer par la chambre d&#39;écoulement. </li><li> Lavez 15 pi SA-tampon dans chaque couloir. Attendre 10 minutes, ou jusqu&#39;à 2 heures. La streptavidine se lie à la biotine lié à la surface-BSA. </li><li> Laver chaque voie trois fois avec 15 pi de tampon BSA pour retirer gratuitement SA, tout en continuant de bloquer la surface de glissement. </li><li> Laver chaque voie avec 15 ul de tampon α-caséine. α-caséine permet en outre de bloquer liaison non spécifique de la kinésine et des microtubules dans le verre. </li><li> Diluer kinésine à 10 nM dans α-caséine tampon et laver chaque voie avec 15 ul de cette kinésine - α-caséine solution. Attendre 15 minutes (ou jusqu&#39;à une heure) de la kinésine biotinylé de se lier spécifiquement à la streptavidine liée à la surface. Les domaines kinésine moteur resteront libres de lier les microtubules. </li><li> Diluer le paclitaxel à 40 pM dans la température ambiante α-caséine tampons, et laver chaque voie avec 15 ul de cette solution pour laver la kinésine libre et de pré-charger chaque chambre d&#39;écoulement avec une solution de paclitaxel pour éviter de dépolymérisation des microtubules. Froide dépolymérise également microtubules, alors assurez-vous que ces étapes se produisent à température ambiante avec salle de temre des solutions. </li><li> Diluer marqué par fluorescence microtubules 1:100-1:1,000 de la fluorescence anti-eau de Javel tampon avec 1 mM d&#39;ATP. Lavez 15 ul dans une voie et d&#39;observer dans les 30 minutes. </li></ol> 4. Collecte des données <ol><li> Observez le glissement des microtubules en utilisant la microscopie à fluorescence, un microscope capable de résoudre fluorophores simples comme une installation commerciale ou maison de construction FRBR est nécessaire 26 (figure 2). Les microtubules devrait être propulsé par les moteurs de kinésine sur le substrat à environ 0,5 mm / s, en fonction de la température. Si le champ de vision du microscope est de 50 um, les microtubules individuels doit être visible pendant environ 100 secondes. Réglez l&#39;intensité lumineuse de telle sorte que les fluorophores simples ne photoblanchiment plus vite que 100 sec. Si l&#39;on utilise une excitation laser, un réglage de puissance de l&#39;ordre de 3-5 mW est approprié. </li><li> Recueillir des séquences d&#39;images pour l&#39;analyse. 600 images à5 Hz (2 min au total) fonctionne bien. Utiliser des séquences assez longtemps que les microtubules traversent l&#39;ensemble du champ de vision. </li></ol> 5. Analyse des données Une routine IDL, <a href="https://www.jove.com/files/ftp_upload/50117/50117IDL_Routines.zip" target="_blank">get_lp.pro</a> , est annexé. Cette routine renvoie une valeur de longueur de persistance basée sur les microtubules glisse dans une séquence d&#39;images donné. Soit exécuter cette routine sur chaque séquence d&#39;images, la modification des paramètres d&#39;intensité en fonction de la configuration de votre microscope particulier, ou procédez comme suit: <ol><li> Suivre chaque fluorophore attaché à un microtubule à créer des trajectoires fluorophores. </li><li> Mélanger tous les trajectoires des fluorophores sur un microtubule donné dans une trajectoire globale des microtubules; répétez pour chaque microtubules dans la séquence d&#39;images (figure 3). </li><li> Calculer l&#39;angle de la tangente à chaque point le long de la trajectoire (figure 4), Et calculer &lt;cos θ s&gt; dans l&#39;équation. 1 en faisant la moyenne du cosinus de la différence d&#39;angle pour chaque paire de points séparés par un chemin de longueur l (figure 5). Trouver la longueur de persistance pour un microtubule ou un groupe de microtubules par &lt;cos montage θ s&gt; à Eq. 1, la pondération des points de données individuels dans l&#39;ajustement par le nombre de valeurs indépendantes du cos θ s qui sont utilisés pour calculer la moyenne. </li></ol>

<ol>
	<li>Hawkins, T., Mirigian, M., Selcuk Yasar, M., Ross, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+microtubules.">Mechanics of microtubules.</a> Journal of biomechanics. 43, 23-30 (2010).</li><li>Pampaloni, 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=Thermal+fluctuations+of+grafted+microtubules+provide+evidence+of+a+length-dependent+persistence+length.">Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length.</a> Proceedings of the national academy of sciences U S A. 103, 10248-10253 (2006).</li><li>Taute, K. M., Pampaloni, F., Frey, E., Florin, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+depart+from+the+wormlike+chain+model.">Microtubule dynamics depart from the wormlike chain model.</a> Physical review letters. 100, 028102 (2008).</li><li>Van den Heuvel, M. G., de Graaff, M. P., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+curvatures+under+perpendicular+electric+forces+reveal+a+low+persistence+length.">Microtubule curvatures under perpendicular electric forces reveal a low persistence length.</a> Proceedings of the national academy of sciences U.S.A. 105, 7941-7946 (2008).</li><li>Gittes, F., Mickey, B., Nettleton, J., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexural+rigidity+of+microtubules+and+actin+filaments+measured+from+thermal+fluctuations+in+shape.">Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape.</a> Journal of cell biology. 120, 923-934 (1993).</li><li>Brangwynne, C. P., 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=Bending+dynamics+of+fluctuating+biopolymers+probed+by+automated+high-resolution+filament+tracking.">Bending dynamics of fluctuating biopolymers probed by automated high-resolution filament tracking.</a> Biophysical journal. 93, 346-359 (2007).</li><li>Cassimeris, L., Gard, D., Tran, P. T., Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XMAP215+is+a+long+thin+molecule+that+does+not+increase+microtubule+stiffness.">XMAP215 is a long thin molecule that does not increase microtubule stiffness.</a> Journal of cell science. 114, 3025-3033 (2001).</li><li>Valdman, D., Atzberger, P. J., Yu, D., Kuei, S., Valentine, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+analysis+methods+for+the+robust+measurement+of+the+flexural+rigidity+of+biopolymers.">Spectral analysis methods for the robust measurement of the flexural rigidity of biopolymers.</a> Biophysical. 102, 1144-1153 (2012).</li><li>van Mameren, J., Vermeulen, K. C., Gittes, F., Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leveraging+single+protein+polymers+to+measure+flexural+rigidity.">Leveraging single protein polymers to measure flexural rigidity.</a> Journal of physical chemistry b. 113, 3837-3844 (2009).</li><li>Hua, W., Chung, J., Gelles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinguishing+inchworm+and+hand-over-hand+processive+kinesin+movement+by+neck+rotation+measurements.">Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements.</a> Science. 295, 844-848 (2002).</li><li>Duke, T., Holy, T. E., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Gliding+assays"+for+motor+proteins:+A+theoretical+analysis.">"Gliding assays" for motor proteins: A theoretical analysis.</a> Physical review letters. 74, 330-333 (1995).</li><li>Mehrbod, M., Mofrad, 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=On+the+significance+of+microtubule+flexural+behavior+in+cytoskeletal+mechanics.">On the significance of microtubule flexural behavior in cytoskeletal mechanics.</a> PLoS one. 6, e25627 (2011).</li><li>Szarama, K. B., Gavara, N., Petralia, R. S., Kelley, M. W., Chadwick, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+changes+in+actin+and+microtubules+underlie+the+developing+surface+mechanical+properties+of+sensory+and+supporting+cells+in+the+mouse+cochlea.">Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea.</a> Development. 139, 2187-2197 (2012).</li><li>Gal, N., Weihs, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Mechanics+and+Activity+of+Breast+Cancer+Cells+Correlate+with+Metastatic+Potential.">Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential.</a> Cell biochemistry and biophysics. , (2012).</li><li>Volokh, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+tensegrity+in+cell+mechanics.">On tensegrity in cell mechanics.</a> Mol. Cell Biomech. 8, 195-214 (2011).</li><li>Kasas, S., Dietler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+measuring+microtubule+stiffness.">Techniques for measuring microtubule stiffness.</a> Current nanoscience. 3, 85-96 (2007).</li><li>Kikumoto, M., Kurachi, M., Tosa, V., Tashiro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexural+rigidity+of+individual+microtubules+measured+by+a+buckling+force+with+optical+traps.">Flexural rigidity of individual microtubules measured by a buckling force with optical traps.</a> Biophysical journal. 90, 1687-1696 (2006).</li><li>Venier, P., Maggs, A. C., Carlier, M. F., Pantaloni, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+microtubule+rigidity+using+hydrodynamic+flow+and+thermal+fluctuations.">Analysis of microtubule rigidity using hydrodynamic flow and thermal fluctuations.</a> Journal of biological chemistry. 269, 13353-13360 (1994).</li><li>van den Heuvel, M. G., Bolhuis, S., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+length+measurements+from+stochastic+single-microtubule+trajectories.">Persistence length measurements from stochastic single-microtubule trajectories.</a> Nano letters. 7, 3138-3144 (2007).</li><li>Phillips, R., Kondev, J., Theriot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physical biology of the cell. , (2008).</li><li>Berliner, E., Young, E. C., Anderson, K., Mahtani, H. K., Gelles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+a+single-headed+kinesin+to+track+parallel+to+microtubule+protofilaments.">Failure of a single-headed kinesin to track parallel to microtubule protofilaments.</a> Nature. 373, 718-721 (1995).</li><li>Anderson, E. K., Martin, D. 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+fluorescent+GTP+analog+as+a+specific,+high-precision+label+of+microtubules.">A fluorescent GTP analog as a specific, high-precision label of microtubules.</a> Biotechniques. 51, 43-48 (2011).</li><li>Hyman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+modified+tubulins.">Preparation of modified tubulins.</a> Methods in enzymology. 196, 478-485 (1991).</li><li>Lee, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curve+reconstruction+from+unorganized+points.">Curve reconstruction from unorganized points.</a> Computer aded geometric design. 17, 161-177 (2000).</li><li>Yildiz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+V+walks+hand-over-hand:+Single+fluorophore+imaging+with+1.5-nm+localization.">Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization.</a> Science. 300, 2061-2065 (2003).</li><li>Friedman, L. J., Chung, J., Gelles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viewing+dynamic+assembly+of+molecular+complexes+by+multi-wavelength+single-molecule+fluorescence.">Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence.</a> Biophysical. 91, 1023-1031 (2006).</li><li>Smith, C. S., Joseph, N., Rieger, B., Lidke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+single-molecule+localization+that+achieves+theoretically+minimum+uncertainty.">Fast, single-molecule localization that achieves theoretically minimum uncertainty.</a> Nature Methods. 7, 373-375 (2010).</li><li>Nitzsche, B., Ruhnow, F., Diez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum-dot-assisted+characterization+of+microtubule+rotations+during+cargo+transport.">Quantum-dot-assisted characterization of microtubule rotations during cargo transport.</a> Nature. 3, 552-556 (2008).</li></ol>

Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Les mesures de longueur de persistance sont une bonne caractérisation des propriétés mécaniques des biopolymères individuels. Dans cet article, nous avons décrit une méthode de mesure de la longueur de persistance des microtubules. Comme indiqué dans l&#39;introduction, cette méthode est facilement étendu à l&#39;examen des propriétés mécaniques des microtubules dans une variété de conditions tout simplement en faisant varier les réactifs, la température, la viscosité ou à l&#39;étape finale de l&#3...

The cytoskeleton, a network of biopolymers found in most eukaryotic cells, plays a role in cellular organization, intracellular transport, and cell mechanics. The mechanical characteristics of the biopolymers of the cytoskeleton (primarily actin and microtubules) play a significant role in determining the mechanical characteristics of the cell as a whole12. Since whole cell mechanics can characterize healthy and diseased cells13,14 and is involved in cellular motility15, the mechanical properties of the underlying cytoskeletal components have been an active area of study for the past two decades1.
The flexibility (or stiffness) of biopolymers is characterized by the persistence length, the length of polymer which bends by approximately one radian under thermal fluctuations at ambient temperature. A number of techniques have been developed to measure persistence length16, for example active techniques which involve bending the polymer using hydrodynamic flow, optical traps, or electric fields4,17,18 , and passive techniques which measure the fluctuations of free polymers in solution5,6 . The active measurements, however, require specialized setups to implement known forces on the micrometer scale, and the free-fluctuation measurements can be challenging due to diffusion out of the plane of focus of the microscope used.
In this article, we describe a complementary, passive, technique to measure the persistence length of microtubules, a cytoskeletal polymer. The technique involves gliding assays, which ensure that the polymer always remains in the focal plane19. Moreover, it involves tracking single fluorophores attached permanently to the polymer of interest, so that specific locations along the polymer are well characterized.
A cartoon of the method is shown in Figure 1. Kinesin moves specifically toward the + end of microtubules, so the microtubules in a gliding assay are propelled unidirectionally. The leading end of the microtubule, beyond the last kinesin attached, is free to fluctuate under the thermal forces of the surrounding solution. As the microtubule is propelled forward, the end fluctuates until binding to a new kinesin molecule further along the glass slide freezes in a given fluctuation. Because kinesin attaches microtubules very strongly, the microtubule is constrained to follow the path of the leading end. Therefore, the statistical fluctuations frozen into the microtubule trajectory are the same as the statistical fluctuations of the free end of microtubules11, and can therefore be used to calculate the persistence length according to20
<img alt="Equation 1" fo:content-width="1in" fo:src="/files/ftp_upload/50117/50117eq1highres.jpg" src="/files/ftp_upload/50117/50117eq1.jpg" /> 
where lp is the persistence length of the microtubule, &theta;s is the angle between tangents to the trajectory separated by a contour length s, and &lt;&gt; denotes an average over all pairs of positions separated by a contour length s.
The gliding assay itself uses kinesin biotinylated at the coiled-coil21 specifically bound to the glass slide via a streptavidin-biotin linkage. This attachment ensures that the motor domains are free to bind to and propel microtubules. In order to follow microtubule trajectories, microtubules are sparsely labeled with organic fluorophores22,23 - the labels must be sparse enough that single fluorophores are resolvable using single molecule fluorescence microscopy. Single fluorophores are tracked using image analysis routines written in IDL. The trajectories of each fluorophore bound to a given microtubule are combined into a composite microtubule trajectory automatically24. The tangent angles &theta; to each point along a trajectory are calculated; from these tangent angles the &lt;cos&theta;s&gt; value is calculated for each contour length s. Finally, these data are fit to Eq. 1 in order to extract a persistence length for a given microtubule, or for many microtubules in the same gliding assay.
The method is robust enough to work with microtubules prepared in a wide variety of conditions (with different stabilizing agents or other small molecules bound to the microtubule, with bound microtubule associated proteins (MAPs), or with a variety of viscous solutions). In our lab, the technique has been used to characterize the persistence length of microtubules as a function of length along the microtubules and microtubules with different stabilizing agents. The main restriction is that the microtubules must still support kinesin motility. Since kinesin is a robust motor enzyme, this is a fairly loose restriction. By replacing microtubules with actin and kinesin with a myosin family enzyme, the persistence length of actin can be measured using the same technique.

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 <td>imidazole</td>
 <td>Sigma-Aldrich</td>
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 <td>potassium chloride</td>
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 <td>magnesium chloride</td>
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 <td>BSA</td>
 <td>Calbiochem</td>
 <td>126615</td>
 <td></td>
 </tr>
 <tr>
 <td>biotinylated BSA</td>
 <td>Thermo Scientific</td>
 <td>29130</td>
 <td></td>
 </tr>
 <tr>
 <td>&alpha;-casein</td>
 <td>Sigma-Aldrich</td>
 <td>C6780 </td>
 <td></td>
 </tr>
 <tr>
 <td>streptavidin</td>
 <td>Thermo Scientific</td>
 <td>21125</td>
 <td></td>
 </tr>
 <tr>
 <td>dithiothreitol</td>
 <td>Sigma-Aldrich</td>
 <td>D0632 </td>
 <td></td>
 </tr>
 <tr>
 <td>paclitaxel</td>
 <td>LC Laboratories</td>
 <td>P-9600</td>
 <td></td>
 </tr>
 <tr>
 <td>glucose oxidase</td>
 <td>Sigma-Aldrich</td>
 <td>G2133</td>
 <td></td>
 </tr>
 <tr>
 <td>catalase</td>
 <td>Sigma-Aldrich</td>
 <td>C100</td>
 <td></td>
 </tr>
 <tr>
 <td>glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383 </td>
 <td></td>
 </tr>
 <tr>
 <td>2-mercaptoethanol</td>
 <td>Sigma-Aldrich</td>
 <td>M3148 </td>
 <td>Toxic. Buy small amount.</td>
 </tr>
 <tr>
 <td>24X60 mm No. 1 1/2 cover glass</td>
 <td>VWR</td>
 <td>48393-252</td>
 <td></td>
 </tr>
 <tr>
 <td>22X22 mm No. 1 cover glass</td>
 <td>Gold Seal</td>
 <td>3306</td>
 <td></td>
 </tr>
 <tr>
 <td>High Vacuum Grease</td>
 <td>Dow-Corning</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>TIRF microscope</td>
 <td>many</td>
 <td>NA</td>
 <td>The TIRF microscope used in this method was home-made.</td>
 </tr>
 <tr>
 <td>IDL (software)</td>
 <td>Exelis</td>
 <td>NA</td>
 <td>Could substitute MATLAB, ImageJ, or other image analysis software.</td>
 </tr>
	 </tbody>
 </table>

flexural rigidity measurements of biopolymers using gliding assays

Les microtubules sont des polymères du cytosquelette qui jouent un rôle dans la division cellulaire, mécanique cellulaire et le transport intracellulaire. Chacune de ces fonctions nécessite microtubules qui sont raides et droites assez pour couvrir une fraction significative du diamètre cellulaire. En conséquence, la longueur de persistance des microtubules, une mesure de rigidité, a été activement étudiée pour les deux dernières décennies 1. Néanmoins, des questions restent ouvertes: microtubules courts sont 10-50 fois moins rigide que les microtubules longues 2-4, et même microtubules longs ont mesuré la longueur de persistance qui varient d&#39;un ordre de grandeur 5-9. Ici, nous présentons une méthode pour mesurer la longueur de persistance des microtubules. La méthode est basée sur une analyse axée sur la kinésine microtubules glisse 10. En combinant rares marquage fluorescent des microtubules individuels avec suivi de particule unique de fluorophores individuelles attachées à des microtubules, le Glidintrajectoires g de microtubules simples sont suivis du nanomètre-niveau de précision. La longueur de persistance de la trajectoire est la même que la longueur de persistance de l&#39;microtubules dans les conditions utilisées 11. Un sous-programme de suivi automatisé est utilisé pour créer des trajectoires des microtubules à partir de fluorophores liés aux microtubules individuels, et la longueur de persistance de cette trajectoire est calculée à l&#39;aide des routines écrites en langage IDL. Cette technique est rapidement réalisable, et capable de mesurer la longueur de persistance de 100 microtubules dans un jour de l&#39;expérimentation. La méthode peut être étendue pour mesurer la longueur de persistance sous une variété de conditions, y compris la durée de persistance en fonction de la longueur le long des microtubules. En outre, les routines d&#39;analyse utilisés peuvent être étendus à la myosine à base de tests effet de glisse, pour mesurer la longueur de persistance des filaments d&#39;actine ainsi.

A snapshot from a gliding assay is shown in Figure 2. A good microtubule density is 1-10 microtubules per field of view; substantially more will result in mistracking as microtubules cross each other. A plot of the 11 microtubule trajectories from the gliding assay in Figure 2 is shown in Figure 3. Typical trajectories are 10 to 30 &mu;m long; some trajectories have gaps where one microtubule crosses another. These trajectories may be discarded from analysis....

Watch this Scientific Journal Video about Flexural Rigidity Measurements of Biopolymers Using Gliding Assays at JoVE.com

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Procédé pour mesurer la longueur de persistance ou la rigidité en flexion de biopolymères est décrit. La méthode utilise une kinésine axée sur les microtubules glisse test pour déterminer expérimentalement la longueur de persistance des microtubules individuels et est adaptable à l&#39;actine des essais à base de glisse.

Mesures rigidité en flexion des biopolymères en utilisant des essais de vol à voile

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

flexural-rigidity-measurements-of-biopolymers-using-gliding-assays

Research

JoVE Journal

Bioengineering

10.7K vues.  Lawrence University. Procédé pour mesurer la longueur de persistance ou la rigidité en flexion de biopolymères est décrit. La méthode utilise une kinésine axée sur les microtubules glisse test pour déterminer expérimentalement la longueur de persistance des microtubules individuels et est adaptable à l'actine des essais à base de glisse.

Vidéo: Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Développement de ovaire de hamster chinois-hôte dosages de protéines cellulaires utilisant la technologie acoustique microparticules membranaires

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Recherche

Bio-ingénierie

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexée unique molécule mesures Protéolyse forcer l&#39;utilisation de pinces magnétiques

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particules vélocimétrie par image de profil de vitesse pour mesures de débits Micro sang

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Mesure longitudinale de la matrice extracellulaire rigidité de tumeur modèles 3D utilisant Microrhéologie de particules de suivi

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Perles agrégation essais pour la caractérisation de molécules d&#39;adhésion cellulaire putatif

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Enchevêtrement moléculaire et Electrospinnability de biopolymères

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Méthodes pour l&#39;auto-intégration de Megamolecular biopolymères sur le séchage à l&#39;air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

Mesures d’entrée de puissance dans des bioréacteurs brassées à l’échelle du laboratoire

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Un système de test en flexion millimètre pour mesurer les propriétés mécaniques des Spicules d’éponges marines

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.