This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

<ol><li>Levental, I., Georges, P. C., Janmey, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Soft+biological+materials+and+their+impact+on+cell+function%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">Soft biological materials and their impact on cell function.</a> Soft Matter. 3, 299-306 (2007).</li>
<li>Minton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanotransduction%3A+A+stiff+response%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Mechanotransduction: A stiff response.</a> Nature Reviews Molecular Cell Biology. 15 (8), 500-500 (2014).</li>
<li>Yeung, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+substrate+stiffness+on+cell+morphology%2C+cytoskeletal+structure%2C+and+adhesion%5BTitle%5D)+AND+%22Cell+motility+and+the+cytoskeleton%22%5BJournal%5D)">Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.</a> Cell motility and the cytoskeleton. 60 (1), 24-34 (2005).</li>
<li>Watt, F. M., Huck, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+extracellular+matrix+in+regulating+stem+cell+fate%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Role of the extracellular matrix in regulating stem cell fate.</a> Nature Reviews Molecular Cell Biology. 14 (8), 467-473 (2013).</li>
<li>Zustiak, S., Nossal, R., Sackett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiwell+stiffness+assay+for+the+study+of+cell+responsiveness+to+cytotoxic+drugs%5BTitle%5D)+AND+%22Biotechnology+and+bioengineering%22%5BJournal%5D)">Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs.</a> Biotechnology and bioengineering. 111 (2), (2014).</li>
<li>Mih, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multiwell+platform+for+studying+stiffness-dependent+cell+biology%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A multiwell platform for studying stiffness-dependent cell biology.</a> PLoS One. 6 (5), e19929(2011).</li>
<li>Sunyer, R., Jin, A. J., Nossal, R., Sackett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabrication+of+hydrogels+with+steep+stiffness+gradients+for+studying+cell+mechanical+response%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Fabrication of hydrogels with steep stiffness gradients for studying cell mechanical response.</a> PloS one. 7 (10), e46107(2012).</li>
<li>Herrick, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PEG-phosphorylcholine+hydrogels+as+tunable+and+versatile+platforms+for+mechanobiology%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">PEG-phosphorylcholine hydrogels as tunable and versatile platforms for mechanobiology.</a> Biomacromolecules. 14 (7), 2294-2304 (2013).</li>
<li>Tokuda, E. Y., Leight, J. L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+matrix+elasticity+with+PEG+hydrogels+to+study+melanoma+drug+responsiveness%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Modulation of matrix elasticity with PEG hydrogels to study melanoma drug responsiveness.</a> Biomaterials. 35 (14), 4310-4318 (2014).</li>
<li>Feng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Substrate+stiffness+influences+the+outcome+of+antitumor+drug+screening+in+vitro%5BTitle%5D)+AND+%22Clinical+hemorheology+and+microcirculation%22%5BJournal%5D)">Substrate stiffness influences the outcome of antitumor drug screening in vitro.</a> Clinical hemorheology and microcirculation. 55 (1), 121-131 (2013).</li>
<li>Ramamoorthi, K., Hara, J., Ito, C., Asuri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Three-Dimensional+Matrix+Stiffness+in+Regulating+the+Response+of+Human+Neural+Cells+to+Toxins%5BTitle%5D)+AND+%22Cellular+and+Molecular+Bioengineering%22%5BJournal%5D)">Role of Three-Dimensional Matrix Stiffness in Regulating the Response of Human Neural Cells to Toxins.</a> Cellular and Molecular Bioengineering. 7 (2), 1-7 (2014).</li>
<li>Tilghman, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS one. 5 (9), e12905(2010).</li>
<li>Gobaa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artificial+niche+microarrays+for+probing+single+stem+cell+fate+in+high+throughput%5BTitle%5D)+AND+%22Nature+methods%22%5BJournal%5D)">Artificial niche microarrays for probing single stem cell fate in high throughput.</a> Nature methods. 8 (11), 949-955 (2011).</li>
<li>Kumachev, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+generation+of+hydrogel+microbeads+with+varying+elasticity+for+cell+encapsulation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">High-throughput generation of hydrogel microbeads with varying elasticity for cell encapsulation.</a> Biomaterials. 32 (6), 1477-1483 (2011).</li>
<li>Fu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+regulation+of+cell+function+with+geometrically+modulated+elastomeric+substrates%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Mechanical regulation of cell function with geometrically modulated elastomeric substrates.</a> Nature Methods. 7 (9), 733-736 (2010).</li>
<li>Pelham, R. J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc Natl Acad Sci U S A. 94 (25), 13661-13665 (1997).</li>
<li>Tse, J. R., Engler, A. J., et al. Preparation of hydrogel substrates with tunable mechanical properties. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTse%2C+JR+%22Current+protocols+in+cell+biology+%2F+editorial+board%2C+Juan+S.+Bonifacino+...+%5Bet+al.%5D%22">Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]</a>. 10 (Unit 10 16), (2010).</li>
<li>Yeung, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+substrate+stiffness+on+cell+morphology%2C+cytoskeletal+structure%2C+and+adhesion%5BTitle%5D)+AND+%22Cell+motility+and+the+cytoskeleton%22%5BJournal%5D)">Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.</a> Cell motility and the cytoskeleton. 60 (1), 24-34 (2005).</li>
<li>Lo, C. -M., Wang, H. -B., Dembo, M., Wang, Y. -l <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+movement+is+guided+by+the+rigidity+of+the+substrate%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">Cell movement is guided by the rigidity of the substrate.</a> Biophysical journal. 79 (1), 144-152 (2000).</li>
<li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+elasticity+directs+stem+cell+lineage+specification%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li>
<li>Discher, D. E., Janmey, P., Wang, Y. -l <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310 (5751), 1139-1143 (2005).</li>
<li>Young, D. A., Choi, Y. S., Engler, A. J., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+adipogenesis+of+adult+adipose-derived+stem+cells+using+substrates+that+mimic+the+stiffness+of+adipose+tissue%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue.</a> Biomaterials. 34 (34), 8581-8588 (2013).</li>
<li>Semler, E. J., Lancin, P. A., Dasgupta, A., Moghe, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+hepatocellular+morphogenesis+and+function+via+ligand-presenting+hydrogels+with+graded+mechanical+compliance%5BTitle%5D)+AND+%22Biotechnology+Bioengineering%22%5BJournal%5D)">Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance.</a> Biotechnology Bioengineering. 89 (3), 296-307 (2005).</li>
<li>Reinhart-King, C. A., Dembo, M., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-cell+mechanical+communication+through+compliant+substrates%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">Cell-cell mechanical communication through compliant substrates.</a> Biophysical journal. 95 (12), 6044-6051 (2008).</li>
<li>Fischer, R. S., Myers, K. A., Gardel, M. L., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stiffness-controlled+three-dimensional+extracellular+matrices+for+high-resolution+imaging+of+cell+behavior%5BTitle%5D)+AND+%22Nature+protocols%22%5BJournal%5D)">Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior.</a> Nature protocols. 7 (11), 2056-2066 (2012).</li>
<li>Quinlan, A. M., Billiar, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigating+the+role+of+substrate+stiffness+in+the+persistence+of+valvular+interstitial+cell+activation%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation.</a> Journal of Biomedical Materials Research Part A. 100 (9), 2474-2482 (2012).</li>
<li>Zustiak, S. P., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrolytically+degradable+poly%28ethylene+glycol%29+hydrogel+scaffolds+with+tunable+degradation+and+mechanical+properties%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties.</a> Biomacromolecules. 11 (5), 1348-1357 (2010).</li>
<li>Chrambach, A., Rodbard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyacrylamide+gel+electrophoresis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Polyacrylamide gel electrophoresis.</a> Science. 172 (3982), 440-451 (1971).</li>
<li>Lin, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanosensing+of+substrate+thickness%5BTitle%5D)+AND+%22Physical+review.+E%2C+Statistical%2C+nonlinear%2C+and+soft+matter+physics%22%5BJournal%5D)">Mechanosensing of substrate thickness.</a> Physical review. E, Statistical, nonlinear, and soft matter physics. 82 (4), 041918(2010).</li>
<li>Chrambach, A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChrambach%2C+A+%22The+Practice+of+Quantitative+Gel+Electrophoresis%22">The Practice of Quantitative Gel Electrophoresis</a>. , (1985).</li>
<li>Sagvolden, G., Giaever, I., Pettersen, E. O., Feder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+adhesion+force+microscopy%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Cell adhesion force microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (2), 471-476 (1999).</li>
<li>Javaherian, S., Li, K. J., McGuigan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+and+rapid+method+for+generating+patterned+co-cultures+with+stable+interfaces%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">A simple and rapid method for generating patterned co-cultures with stable interfaces.</a> BioTechniques. 55 (1), 21-26 (2013).</li>
<li>Tarone, G., Galetto, G., Prat, M., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+surface+molecules+and+fibronectin-mediated+cell+adhesion%3A+effect+of+proteolytic+digestion+of+membrane+proteins%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.</a> The Journal of cell biology. 94 (1), 179-186 (1982).</li>
<li>Trujillo, V., Kim, J., Hayward, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creasing+instability+of+surface-attached+hydrogels%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">Creasing instability of surface-attached hydrogels.</a> Soft Matter. 4 (3), 564-569 (2008).</li>
<li>Saha, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+creasing+instability+of+soft+polyacrylamide+cell+culture+substrates%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">Surface creasing instability of soft polyacrylamide cell culture substrates.</a> Biophysical journal. 99 (12), L94-L96 (2010).</li>
<li>Buxboim, A., Rajagopal, K., Andre’EX, B., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+deeply+cells+feel%3A+methods+for+thin+gels%5BTitle%5D)+AND+%22Journal+of+Physics%3A+Condensed+Matter%22%5BJournal%5D)">How deeply cells feel: methods for thin gels.</a> Journal of Physics: Condensed Matter. 22 (19), 194116(2010).</li>
<li>Merkel, R., Kirchgessner, N., Cesa, C. M., Hoffmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+force+microscopy+on+elastic+layers+of+finite+thickness%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">Cell force microscopy on elastic layers of finite thickness.</a> Biophysical journal. 93 (9), 3314-3323 (2007).</li>
</ol>

The authors have nothing to disclose.

ג&#39;ל polyacrylamide, שפותח במקור עבור אלקטרופורזה, 28 המשמשים כיום באופן שיגרתי כמו מצעי תרבית תאים כדי לחקור את ההשפעות של מצע נוקשות על מורפולוגיה של תאים, תנועתיות, ותקשורת 3,24,29 בין מאפייני תא אחרים. Polyacrylamide מאפשרת מניפולציה של מצע נוקשות להקיף את הנוקשות של כ?...

רוב הרקמות בגוף הן חומרי viscoelastic רכים עם מודול יאנג הנע בין 0.1 kPa למוח עד 100 kPa לסחוס רך, עדיין, רובם במחקר בתאים במבחנה מתבצע על קלקר תרבית רקמה (TCP) שבו יש מודול של ~ 1-GPA . 1 התאמה נוקשות זו מאוד משפיעה על הדרך בה תאים מגיבים לסביבתם. גוף הולך וגדל של מחקר הוא בכך מסור להבהרת ההשפעה של מצע נוקשות על גורלם של סוגי תאים שונים, 2,3 כולל תאי גזע. 4 כתוצאה מכך, הידרוג מרובה פותחו כדי לסייע בהבנה של תא נוקשות תלויה הביולוגיה כולל polyacrylamide (PA), פוליאתילן גליקול 5-7 (PEG), 8,9 polydimethylsiloxane (PDMS), 10 ואלגינט. 11 בעוד ההוכחה לכך שיש מצע נוקשות השפעה מהותית על גורל תא גדלה, רוב המחקרים שנערכו ב בקנה מידה קטנה עם מספר קטן של samples. מחקרים שיטתיים, רב-ממדיים על ההשפעה של מצע נוקשות למערך של סוגי תאים או תנאים סביבתיים הם נדירים. 12 מספר טכנולוגיות הידרוג&#39;ל תפוקה גבוהה מבטיחות פותחו, כולל microarrays מבוסס PEG, 13 מכשירים microfluidic לייצור microbeads agarose הידרוג&#39;ל, 14 או מיקרו וננו-מוטות שבו קשיחות היא מווסת על ידי הקוטר והגובה של microrods. 15 עם זאת , הטכנולוגיות להכנת מצעים כאלה הם מתוחכמים וזמינים למספר מוגבל של מעבדות. מחקרים רבים מעורבים תגובות תא נוקשות מווסתת מנצל polyacrylamide ג&#39;לי (PA) שהם לא רק זולים ופשוטים ליישום, אלא גם להציג מגוון רלוונטי מבחינה פיזיולוגית של מודול יאנג, כלומר 0.3 -. 300 kPa 16-22 עם זאת, קיימים שיטות לפברק הרשות הפלסטינית ג &#39;לים תרבית תאים הם עבודה אינטנסיבית וכתוצאה מכך הכנהared בקבוצות קטנות. חלק מהקשיים הכרוכים בהכנה של ג&#39;לי הרשות הפלסטינית כמו מצעי תאי גזע מהדרישה שג&#39;לים צריך להיות מוכן: 1) בהעדר החמצן כדי לאפשר פילמור מלא, 2) עם משטח שטוח וחלק להתיר תא אחיד התקשרות והפצה, ו 3) באופן קבוע המודבק בתחתית של צלחת תרבית תאים כדי למנוע צף. כמה קבוצות ניסו לייצר ג&#39;לי רשות לתרבית תאים בקבוצות גדולות. Semler et al. גיליונות עבים מוכנים של ג&#39;לי הרשות הפלסטינית שהיו אז &quot;לחתוך&quot; עם ניקוב חורים והניחו לתוך 96-גם צלחות. 23 עם זאת, שיטה זו מוגבלת לג&#39;לים נוקשה, כלומר,&gt; 1 kPa במודול יאנג, כי רך יותר ג &#39;הם &quot;דביק&quot;, קשה לחתוך, ונפגע בקלות. MIH et al. פיתח טכניקה מתוחכמת יותר המאפשרת לג&#39;לים להיות polymerized ישירות בצלחת multiwell זכוכית תחתונה. <sעד&gt; 6 זו הושגה על ידי שפיכת פתרונות ג&#39;ל לצלחות זכוכית תחתונה פונקציונליות ויצירת ג&#39;לים על ידי &quot;הרבדה&quot; אותם עם מערך coverglass המותאם אישית. 6 למרות שהשפעות קצה מבטיחות מאוד, קלות עדיין נצפו עם טכניקה זו. בנוסף, הטכניקה דורשת מערך אישית מעוצב לא נגיש באופן מיידי למעבדות רבות, כמו גם צלחות multiwell זכוכית תחתונה יקרות. מאמר זה מתאר דרך פשוטה וזולה כדי להרכיב ג&#39;לי הרשות הפלסטינית בצלחת multiwell שאפשר לאמץ בקלות על ידי כל מעבדה. כאן, תמיכת פלסטיק גמישה מנוצלת, שבו יש שני צדדים - אחד הידרופובי, שהוא דוחה לג&#39;לי הרשות הפלסטינית, ואחד הידרופילי, שנקשר קוולנטית ג&#39;ל הרשות הפלסטינית על תצהיר. ברגע גיליונות ג&#39;ל הרשות הפלסטינית מופקדים וקבוע מודבקים תמיכת פלסטיק הגמישה, זה מאפשר טיפול בג&#39;לים של כל עובי או נוקשות וחיתוכם לכל צורה רצויה. appr זהאוח לא רק מייצר מותאם אישית &#39;coverslips&#39; הפלסטיק בגדלים לא אחר זמין מסחרי, אבל גם מונע את הצורך לטיפול מקדים משטחי זכוכית, או coverslips זכוכית או בארות של צלחות multiwell זכוכית תחתונה יקרות, עם פתרון הרשות הפלסטינית מחייב, שהוא מייגע וצעד זמן רב. לבסוף, ניתן להכין גיליונות ג&#39;לי הרשות הפלסטינית אחידים בקבוצות גדולות ומאוחסנים דה-hydrated במשך כמה חודשים. לסיכום, assay שהוצג כאן הוא שיפור פני שיטות קיימות במספר היבטים. ראשית, התהליך של הרכבה צלחת multiwell הוא יעיל, ואת העלות הכוללת של חומרים הדרושים היא נמוכה. שנית, הידרוג מיוצר בקבוצות גדולות בסרט ג&#39;ל הומוגנית אחת. לבסוף, רק חומרים שהם זמינים מסחריים נדרשים. השירות של assay מודגם על ידי לחקור את ההשפעה של מצע נוקשות על מורפולוגיה של תאים ומתפשטים באזור.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>40% Acrylamide</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>2% Bis-acrylamide</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Bio-Rad</td><td>161-07000</td><td></td></tr><tr><td>TEMED</td><td>Sigma Aldrich</td><td>T9281</td><td></td></tr><tr><td>Sulfo-SANPAH</td><td>Thermo Scientific</td><td>22589</td><td></td></tr><tr><td>Collagen Type 1, from Rat tail, 3.68 mg/ml</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>Hydrophobic solution &amp;#8212; Repel Silane</td><td>GE Healthcare Bio-Sciences</td><td>17-1332-01</td><td></td></tr><tr><td>PBS (1x), pH 7.4</td><td>HyClone</td><td>SH30256.01</td><td></td></tr><tr><td>Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td><td>Elsworth Adhesives</td><td>3097358-1004</td><td></td></tr><tr><td>Tyrpsin/EDTA (10x)</td><td>Sigma Aldrich</td><td>44174</td><td></td></tr><tr><td>RPMI-1640 Medium (1x)</td><td>HyClone</td><td>SH30027-02</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30073-03</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>MP Biomedicals</td><td>1670046</td><td></td></tr><tr><td>Detergent: Triton-X</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Formaldehyde 37% Solution</td><td>Sigma Aldrich</td><td>F1635</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>BSA-based cell adhesion blocking kit &amp;#8212; ECM Cell Adhesion Array Kit</td><td>Chemicon International</td><td>ECM540</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Disposable lab equipment&lt;/strong&gt;</td></tr><tr><td>flexible plastic support &amp;#8212; GelBond PAG Film for Polyacrylamide Gels</td><td>GE Healthcare Bio-Sciences</td><td>309819</td><td></td></tr><tr><td>Glass Plates</td><td>Slumpys</td><td>GBS4100SFSL</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Fisher Scientific</td><td>3181345107</td><td></td></tr><tr><td>15 ml conicals tubes</td><td>FALCON</td><td>352097</td><td></td></tr><tr><td>Micro centrifuge tubes</td><td>Fisher Scientific</td><td>2 ml: 02681258</td><td></td></tr><tr><td>96-well plate (flat bottom)</td><td>Fisher Scientific</td><td>12565501</td><td></td></tr><tr><td>Disposable Pipettes (1&amp;nbsp;ml, 2&amp;nbsp;ml, 5&amp;nbsp;ml, 10&amp;nbsp;ml, 25&amp;nbsp;ml, 50&amp;nbsp;ml)</td><td>Fisher Scientific</td><td>1 ml: 13-678-11B, 2&amp;nbsp;ml: 05214038, 5&amp;nbsp;ml (FALCON): 357529, 10&amp;nbsp;ml: 13-678-11E, 25&amp;nbsp;ml: 13-678-11, 50&amp;nbsp;ml: 13-678-11F</td><td></td></tr><tr><td>Glass Transfer Pipettes</td><td>Fisher Scientific</td><td>5 3/4&quot;: 1367820A, 9&quot;:136786B</td><td></td></tr><tr><td>Pipette Tips (1-200&amp;nbsp;&amp;#956;l, 101-1000&amp;nbsp;&amp;#956;l)</td><td>Fisher Scientific</td><td>2707509</td><td></td></tr><tr><td>Plastic Standard Disposable Transfer Pipettes</td><td>Fisher Scientific</td><td>13-711-9D</td><td></td></tr><tr><td>Parafilm</td><td>PARAFILM&amp;nbsp;</td><td>PM992</td><td></td></tr><tr><td>Powder Free Examination Gloves</td><td>Quest</td><td>92897</td><td></td></tr><tr><td>Silicone spacers &amp;#8212; Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td><td>Grace Bio-Labs</td><td>JTR-S-0.5</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Large/non-disposable lab equipment&lt;/strong&gt;</td></tr><tr><td>Light and Flourescent Microscope (Axiovert 200M)</td><td>Zeiss</td><td>3820005619</td><td></td></tr><tr><td>Microscope Software</td><td>Zeiss</td><td>AxioVision Rel. 4.8.2</td><td></td></tr><tr><td>UV oven</td><td>UVITRON</td><td>UV1080</td><td></td></tr><tr><td>Vacuum chamber/degasser</td><td>BelArt</td><td>999320237</td><td></td></tr><tr><td>Vacuum pump for degasser</td><td>KNF Lab</td><td>5097482</td><td></td></tr><tr><td>Tissue Culture Hood</td><td>NUAIRE</td><td>NU-425-600</td><td></td></tr><tr><td>Chemical Fume Hood</td><td>KEWAUNEE</td><td>99151</td><td></td></tr><tr><td>Inverted Microscope (Axiovert 25)</td><td>Zeiss</td><td>663526</td><td></td></tr><tr><td>Incubator</td><td>NUAIRE</td><td>NU-8500</td><td></td></tr><tr><td>Pipette Aid</td><td>Drummond Scientific Co.</td><td>P-76864</td><td></td></tr><tr><td>Hemacytometer</td><td>Bright-Line</td><td>383684</td><td></td></tr></tbody></table>

simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Polyacrylamide הידרוג (PA) נמצא בשימוש נרחב כדי לבדוק את תגובות תא נוקשות תלויה. 17,24 על ידי ערבוב ריכוזים שונים של acrylamide () וbis-acrylamide (B) אחד יכול לעשות ג&#39;לי הרשות הפלסטינית כי היקף מגוון הנוקשות של רוב הרקמות הרכות ב הגוף - 0.3 -.. 300 מודולוס של kPa הצעיר 1 עם זאת, הכנת ג&#39;?...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Multiwell הנוקשות Assay לחקר תגובות תא הנוקשת תלויות מבוסס polyacrylamide פשוטה

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.7K Views.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Video: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

הכנת מטריצות תלונה לכימות התכווצות נייד

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

שאיפת micropipette של תאי מצע מצורפים להערכת נוקשות סלולריות

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

מדידת התכונות המכאניות של תאי חיים באמצעות מיקרוסקופ כוח אטומי

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

ג&#39;לים polyacrylamide לInvadopodia וגרירת חיל מבחני בתאי הסרטן

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

Medicine

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

שיחקרו את ההשפעות של מטריקס נוקשות על פונקציה נייד באמצעות Acrylamide מבוססי הידרוג

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Biology

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

מערכת O9-1/הידרוג'ל ממוטבת לחקר אותות מכניים בתאי ציצית עצבית

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Developmental Biology

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

מידה נוקשות של סובסטרטים סיליקון רך Mechanobiology מחקרים באמצעות מיקרוסקופ זריחה Widefield

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Assay מבוססי לזיהוי עיצוב רב טוב ללמוד את ההשפעה של מטריצה חוץ-תאית נוקשות על זיהום חיידקי של תאים חסיד

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Immunology and Infection

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

מערכת בודדת של תא להערכת בקרה מכנית של התנועה הסלולרית ואירועי איתות קשורים

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

Source: Bastounis, E. E. et al., <a href="https://app.jove.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp.&#160;(2018)
This video showcases quantitative time-lapse microscopy for evaluating extracellular matrix stiffness-dependent Listeria monocytogenes dissemination through endothelial cells. A softer hydrogel facilitates faster bacterial dissemination through endothelial cells compared to stiffer counterparts, highlighting the influence of matrix stiffness on bacterial dissemination.

מיקרוסקופ כמותי בהילוך מהיר להערכת הפצת חיידקים חוץ-תאיים תלויי קשיחות

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

	Dissolve ammonium persulfate (APS) in distilled ultrapure water to ...