Name: a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex
Uploaded: 2017-04-11T12:30:00
Description: Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

עבודה זו נתמכה על ידי תומאס פ וקייט מילר Jeffress Memoria Trust (עד דצמבר) ו- NIH להעניק R35GM119617 (עד דצמבר). הדמית מיקרוסקופ confocal בוצעה לעבר Core אפיון ננו VCU (NCC) המתקן.

1. השג Nesprin-2G חיישן דנ&quot;א האחר פלסמיד דנ&quot;א <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השג Nesprin-2G TS (חיישן המתח), Nesprin-2G HL (ללא ראש) מלאה, mTFP1, ונוס, ו TSmod ממקור מסחרי. הפץ כל פלסמידים DNA ולטהר אותם באמצעות זני חיידק סטנדרטיים, כגון-α DH5, כפי שתואר לעיל <sup...

<ol>
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Biochem. 21 (5-6), 489-498 (2008).</li><li>Kardash, E., Bandemer, J., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+activity+in+live+embryos+using+fluorescence+resonance+energy+transfer+biosensors.">Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors.</a> Nat Protoc. 6 (12), 1835-1846 (2011).</li><li>Vaziri, A., Mofrad, M. R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+deformation+of+the+nucleus+in+micropipette+aspiration+experiment.">Mechanics and deformation of the nucleus in micropipette aspiration experiment.</a> J Biomech. 40 (9), 2053-2062 (2007).</li><li>Wang, N., Naruse, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+behavior+in+living+cells+consistent+with+the+tensegrity+model.">Mechanical behavior in living cells consistent with the tensegrity model.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7765-7770 (2001).</li><li>Nagayama, K., Yahiro, Y., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+stabilize+the+position+of+intranuclear+DNA+through+mechanical+connection+with+the+nucleus+in+vascular+smooth+muscle+cells.">Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells.</a> FEBS letters. 585 (24), 3992-3997 (2011).</li><li>Luxton, G. W. G., Gomes, E. R., Folker, E. S., Vintinner, E., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+arrays+of+nuclear+envelope+proteins+harness+retrograde+actin+flow+for+nuclear+movement.">Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement.</a> Science. 329 (5994), 956-959 (2010).</li><li>Chen, Y., Mauldin, J. P., Day, R. N., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+spectral+FRET+imaging+microscopy+for+monitoring+nuclear+protein+interactions.">Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions.</a> J Microsc. 228, 139-152 (2007).</li><li>Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. 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שיטה והדגמה של הדמיה תא חי של מתח מכני על פני Nesprin-2G, חלבון במתחם LINC גרעיני, היה שתוארו לעיל. לפני עבודה זו, טכניקות שונות, כגון שאיפת micropipette, מגנט חרוז cytometry, ליזר-אבלציה מיקרוסקופי, שמשו ליישם עומס על גרעין התא כדי למדוד תכונות החומר בתפזורת שלה 16, <sup c...

חיל-רגיש, חיישני סריג מקודדים גנטיים צמחו לאחרונה ככלי חשוב למדידת כוחות מבוסס מתיחה בתאים חיים, ומספקים תובנות איך מכני כוחות מוחלים על פני חלבוני 1, 2, 3, 4. באמצעות כלים אלה, חוקרים יכולים דימוי הלא פולשני כוחות תאיים בתאים חיים באמצעות מיקרוסקופי פלורסנט קונבנציונליים. חיישנים אלה מורכבים-זוג סריג (פלורסנט תורם acceptor חלבונים, בתדירות הגבוהה ביותר תורם כחול acceptor הצהוב) המופרדים על ידי פפטיד אלסטי 3. בניגוד C- או N-terminal תיוג, חיישן זה מוכנס לתוך אתר פנימי של חלבון כדי למדוד את הכח המכאני ישודר החלבון, מתנהג כמו מד לחץ מולקולרי. גברת מתח מכאני על פני תוצאות החיישן בעל טווח מוגדל בין-p הסריגאוויר, דבר שגרם לפגיעה סריג 3. כתוצאה מכך, הסריג הוא ביחס הפכו כוח מתיחה. חיישנים פלורסנט מבוסס אלו פותחו עבור חלבונים דבקים מוקדים (vinculin 3 ו תלין 4), חלבוני cytoskeletal (α-actinin 5), וחלבוני צומת תא-תא (E-cadherin 6, 7, VE-cadherin 8, ו PECAM 8). הנפוץ ביותר בשימוש היטב מאופיין ומקשר אלסטי ב חיישנים אלה נקרא TSmod והוא מורכב מרצף חוזר ונשנה של 40 חומצות אמינו, (GPGGA) 8, אשר נגזרו flagelliform חלבון משי עכביש. TSmod הוכח להתנהג כמו היענות ננו-אביב אלסטי ליניארי, עם סריג 1 עד 5 pN בכוח מתיחה 3. באורכים שונים של flagelliform יכולים לשמש כדי לשנות את r הדינמיתAnge של TSmod סריג כוח רגישות 9. בנוסף flagelliform, spectrin חוזרת 5 ו פפטיד כיסוי ראש villin (המכונה HP35) 4 שימשו כמו פפטידים אלסטי בין-זוגות סריג ב חיישנים כוח דומה 4. לבסוף, דו&quot;ח אחרון הראה כי TSmod יכול לשמש גם כדי לזהות כוחות דחיסה 10. אנחנו לאחרונה פיתחה חיישן כוח עבור והמקשר של-שלד התא Nucleo (LINC) חלבון מורכב Nesprin2G באמצעות TSmod מוכנס לתוך חלבון שפותחו בעבר קטועה Nesprin2G המכונה מיני-Nesprin2G (איור 2 ג), אשר מתנהג באופן דומה אנדוגניים Nesprin-2G 11. קומפלקס LINC מכיל חלבונים מרובים מפנים מבחוץ לחלק הפנימי של הגרעין, המקשר את שלד תא ציטופלסמית אל למינה הגרעיני. Nesprin-2G הוא חלבון מבני מחייב הוא אתcytoskeleton אקטין בציטופלסמה וכדי חלבונים SUN במרחב perinuclear. שימוש biosensor שלנו, הצלחנו להראות כי Nesprin-2G כפוף מתח actomyosin התלוי NIH3T3 פיברובלסטים 2. זו הייתה הפעם הראשונה בכח כי נמדד ישירות דרך חלבון במתחם LINC גרעיני, וזה עשוי להיות כלי חשוב להבין את התפקיד של כוח על הגרעין ב mechanobiology. פרוטוקול להלן מספק מתודולוגיה מפורט של אופן השימוש בחיישן כוח Nesprin-2G, כולל את הביטוי של חיישן המתח Nesprin (Nesprin-TS) בתאי יונקים, כמו גם רכישת וניתוח של תמונות סריג של תאים המבטאים Nesprin- TS. באמצעות מיקרוסקופ confocal הפוך מצויד בגלאי ספקטרלי, תיאור של איך למדוד פליטה רגישה סריג באמצעות unmixing רפאי הדמית סריג ratiometric מסופקת. תמונות ratiometric הפלט ניתן להשתמש כדי להפוך קואה יחסיתהשוואות כוח ntitative. בעוד פרוטוקול זה מתמקד בביטוי של Nesprin-TS ב פיברובלסטים, זה ניתנת להתאמה בקלות לתאי יונקים אחרים, כולל שתי שורות תאים תאים ראשוניים. יתר על כן, פרוטוקול זה בכל קשור לרכישת תמונה וניתוח סריג ניתן להתאים בקלות חיישני כוח סריג מבוסס אחרים שפותחו עבור חלבונים אחרים.

<table border="0" cellpadding="0" cellspacing="0" width="1183">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-TS DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68127</td>
			<td style="width:475px;">Retrieve from https://www.addgene.org/68127/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-HL DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68128</td>
			<td>Retrieve from https://www.addgene.org/68128/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mTFP1 DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">54613</td>
			<td>Retrieve from https://www.addgene.org/54613/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mVenus DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">27793</td>
			<td>Retrieve from https://www.addgene.org/27793/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">TSmod DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">26021</td>
			<td>Retrieve from https://www.addgene.org/26021/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Competent Cells</td>
			<td style="width:108px;">Bioline</td>
			<td style="width:119px;">BIO-85026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Liquid LB Media</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10855001</td>
			<td>https://www.thermofisher.com/order/catalog/product/10855001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Solid LB Bacterial Culture Plates</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Ampicillin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Spectrophotometer</td>
			<td style="width:108px;">Biorad</td>
			<td style="width:119px;">273 BR 07335</td>
			<td>SmartSpec Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">quartz cuvette</td>
			<td style="width:108px;">Biorad</td>
			<td align="right" style="width:119px;">1702504</td>
			<td>Cuvette for SmartSpec Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">&#160;DNA isolation kit</td>
			<td style="width:108px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740412.5</td>
			<td>NucleoBond Xtra Midi Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">6-well cell culture dish</td>
			<td style="width:108px;">Falcon-Corning</td>
			<td align="right" style="width:119px;">353046</td>
			<td>Multiwell 6-well Polystrene Culture Dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Dulbecco&#39;s Modified Eagle Medium, (DMEM) cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td>DMEM(1x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16170-078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">reduced serum cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>Reduced Serum Medium, &#34;optimem&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Lipid Carrier Solution</td>
			<td style="width:108px;">invitrogen</td>
			<td style="width:119px;">11668-019</td>
			<td>Lipid Reagent, &#34;Lipofectamine 2000&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">1.5mL sterile plastic tube</td>
			<td style="width:108px;">Denville</td>
			<td style="width:119px;">c2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Trypsin</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>0.25% Trypsin-EDTA (1x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">glass-bottom microscope viewing dish</td>
			<td style="width:108px;">In Vitro Scientific</td>
			<td style="width:119px;">D35-20-1.5-N</td>
			<td>35mm Dish with 20mm Bottom Well #1.5 glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Fibronectin</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">33016015</td>
			<td>fibronectin human protein,plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">14190-144</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">15mL sterile centrifuge tube</td>
			<td style="width:108px;">Greiner bio-one</td>
			<td align="right" style="width:119px;">188261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">swinging rotor centrifuge&#160;</td>
			<td style="width:108px;">Thermo electron</td>
			<td style="width:119px;">Centra CL2</td>
			<td>Swinging rotor thermo electron 236</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">cell culture biosafety hood</td>
			<td style="width:108px;">Forma Scientific</td>
			<td align="right" style="width:119px;">1284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;climate controlled cell culture incubator</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">3596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">inverted LED widefield fluorescent microscope</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">EVOS FL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Clear HEPES buffered imaging media</td>
			<td style="width:108px;">Molecular Probes</td>
			<td style="width:119px;">A14291DJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Fetal bovine Serum</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:483px;">Temperature Controlled-Inverted confocal w/458 and 515nm laser sources&#160;</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:119px;">&#160;LSM 710-w/spectral META detector</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Outgrowth Media</td>
			<td style="width:108px;">Newengland Biolabs</td>
			<td style="width:119px;">B9020s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">NIH 3T3 Fibroblasts</td>
			<td style="width:108px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

בעקבות בפרוטוקול לעיל, פלסמיד דנ&quot;א נרכשה מהמאגר דנ&quot;א להפוך לתאי חיידק. E. coli להביע הדנ&quot;א חיישן נבחרו מתוך LB / צלחות אמפיצילין ו מוגבר במרק LB נוזלי. בעקבות ההגברה של הווקטורים, פלסמידים DNA היו מטוהרים לתוך חיץ טריס-EDTA באמצעות תקן, ערכת ב?...

Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

פרוטוקול באמצעות העברת אנרגיה פורסטר תהודה (סריג) חיישנים ביולוגיים -force למדוד כוחות מכניים ברחבי המכלול הגרעיני LINC

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

The overall goal of this procedure is to determine forces on the linker of the nucleoskeleton cytoskeleton, or LINC complex, through FRET microscopy in living cells via a biosensor designed for the LINC complex protein, nesprin-2G. This method can help answer keys questions in the field of mechanotransduction, such as the forces placed on proteins in the nucleus of living cells, and how these forces change under variant conditions. The main advantage of this technique is its ability to measure very small levels of force on proteins and living cells.Generally, individuals new to this method will struggle with capturing images. Optimizing the microscope laser power and gain to receive a good signal between all experimental groups is difficult. To begin, grow NIH 3T3 fiber blast cells to between 70%and 90%confluence in a six-well cell culture dish in a standard cell culture incubator with temperature and CO2 regulation.In a cell culture hood, remove the medium and rinse each well with approximately 1 milliliter of a reduced serum cell medium. Add 800 microliters of reduced serum cell medium to each well, and place the six-well chamber in the incubator. Then, pipette 700 microliters of reduced serum cell medium into a 1.5 milliliter tube labeled L with 35 microliters of lipid carrier solution to form the lipo mix.Mix the suspension by pipetting. Next, pipette 100 microliters of the reduced serum cell medium into each of six 1.5 milliliter tubes labeled one through six. Pipette the plasma DNA for nesprin tension sensor, nesprin headless zero force control, mTFP1, and venus into tubes one through six, as described in the text protocol.Do not reuse pipette tips when pipetting different types of DNA. Then, pipette 100 microliters of the lipo mix from the L tube into each labeled tube, and mix by repeated pipetting, using a clean pipette for each tube. Incubate the suspension for 10 to 20 minutes.Next, add 200 microliters from each labeled tube to a well of the prepared six-well chamber containing cells. Label the top of each well with the corresponding DNA added. Then, place the six-well chamber in an incubator for four to six hours.Following incubation, aspirate the medium, and add one to two milliliters of reduced serum cell medium to rinse. Aspirate the reduced serum cell medium, and add two milliliters of trypsin to each well before placing the six-well dish in the incubator for five to 15 minutes. While the cells detach in the incubator, coat six glass-bottom viewing dishes with a layer of fibronectin at a concentration of 20 micrograms per milliliter dissolved in phosphate-buffered saline.Allow the dishes to coat the surface in the cell culture hood. Neutralize the trypsin by adding two milliliters of DMEM once the cells are detached. Then, transfer the contents of each well to a labeled 15 milliliter centrifuge tube and spin down at 90 times g for five minutes in a swinging rotor centrifuge.Aspirate the supernatant and re-suspend each cell pellet in 1, 000 microliters of DMEM using a pipette. Aspirate the fibronectin mixture from the glass dishes and pipette 1, 000 microliters of each cell suspension onto the dishes. After the cells settle to the bottom of the glass dishes, add another one milliliter of media to each well.Allow the cells to attach and express sensor in the cell incubator for at least 18 to 24 hours. To verify the transfection efficiency, use a fluorescent microscope equipped with an excitation frequency near 462 nanometers for mTFP1, or 525 nanometers for venus. Use an emission filter centered near 492 nanometers for mTFP1, or 525 nanometers for venus.Roughly 18 to 24 hours after completing the transfection, use an inverted fluorescent microscope to examine the efficiency of transfection by comparing the number of fluorescent cells to the total number of cells in view. If live cells cannot be imaged within 48 hours after transfection, fix the cells in 4%paraformaldehyde for five minutes. Allow cells to express sensor for 24 to 48 hours before fixation.Store the cells in PBS and view after 48 hours. In a cell culture hood, replace the cell medium with imaging medium supplemented with 10%fetal bovine serum. Place the viewing dishes in a temperature controlled confocal microscope stage.Place the glass viewing dish with mTFP1 transfected cells over the oil objective at 60X magnification with a numerical aperture of 1.4. Locate mTFP1-expressing cells in widefield fluorescent mode. With a fluorescent cell in the field of view, select the spectral detection mode and capture the spectral image.Include all frequencies beyond 458 nanometers using 10 nanometer increments. Select a bright fluorescent region on the cell. Add the fluorescent ROI mean as the normalized intensity to the spectral database by clicking Save Spectra to Database.Optimize the laser power and gain such that a good signal to noise ratio is achieved. Settings will vary for different equipment. Repeat the process for venus-expressing cells and remain in spectral mode.Use an excitation frequency of 488 nanometers instead of 458 nanometers. Adjust the power settings as needed, but do not change the emission frequencies. Select a bright fluorescent ROI of around a 20-pixel radius.Add the fluorescent ROI mean as the normalized intensity to the spectral database, by clicking Save Spectra to Database. To capture the unmixed images, first switch the capturing mode to spectral unmixing. Add the spectral fingerprints of venus and mTFP1 to the unmixing channels.Set the excitation laser back to a 458 nanometer argon source. Place the nesprin tension sensor viewing dish above the 60X oil objective. After focusing on a fluorescent cell with sufficiently bright expression, adjust the gain and laser power to optimize the signal to noise ratio.Capture a minimum of 15 to 20 images of nesprin tension sensor cells with relatively similar brightness and avoid excessive pixel saturation. Repeat the image capturing process with nesprin headless cells using identical imaging parameters. A representative 10X magnified image of 3T3 fibroblasts transfected with nesprin tension sensor using a lipid mediated carrier is shown here.Typically, only a fraction of cells are transfected in a given field of view. Shown here is a representative pair of two 3T3 nuclei expressing nesprin-TS in the spectrally unmixed venus and mTFP channels. A merged color image of the two channels is shown in the far right panel.Here is a representative sampling of nesprin-TS masked nuclei from stable expressing MDCK cells on patterned and unpatterned substrates. Ratio images are defined as the ratio of the venus channel divided by the mTFP channel and can be used to make relative quantitative force comparisons. Once mastered, this technique can be done in about eight hours if it's performed properly.While attempting this procedure, it's important to remember to carefully label DNA tubes during the transfection, and keep the imaging parameters constant while capturing spectrally result images. After watching this video, you should have a good understanding of how to transfect cells with the nesprin tension sensor and capture spectrally resolved images to computer FRET index that correlates with force on the tension sensor. Though in this case, a FRET sensor is used for nesprin, it can also be applied to other proteins in the cell where measuring force is desired.

Research

JoVE Journal

Bioengineering

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

הוספת בדיקות ביולוגיות Biosensors סיליקה אופטי באמצעות סוכני צימוד silane

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Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

מחקרים קטנים ורחבים זווית X-Ray פיזור של מקרומולקולות ביולוגיות בפתרון

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

כמותית סריג ניתוח (תהודת העברת אנרגית פורסטר) לקביעת SENP1 פרוטאז קינטיקס

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

עיצוב של Bioreactor טוען מכונות Biaxial להנדסת רקמות

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Biosensors רציומטרי שלמדוד את מצב חיזור מיטוכונדריאלי וה-ATP בתאי שמרי חיים

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

מvoxels לידע: מדריך מעשי לפילוח אלקטרונים מורכבים מיקרוסקופית 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

פרוטוקול ליצירת biofilm Streamer במכשיר microfluidic עם מיקרו עמודים

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

הארה תהודת אנרגיה העבר לחקר שינויי קונפורמציה בחלבונים בממברנה שבאו לידי ביטוי בתאי יונקים

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

בודה מצעים מורכבים תרבות באמצעות רובוטית microcontact הדפסה (R-μCP) והתמר נוקלאופילית סדרתית

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...