המחברים מבקשים להודות לאנקת רוטגרות&#39; וקארינה צימרמן לסיוע טכני. עבודה זו נתמכה על ידי Forschungsgemeinschaft דויטשה (מענק NI 1301/1-1 לפון) ואוניברסיטת גטינגן במרכז רפואי (&quot;פרו Futura&quot; מענק לפון).

<ol>
	<li>Zaccolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+chimeric+fluorescent+proteins+and+fluorescence+resonance+energy+transfer+to+monitor+cellular+responses.">Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses.</a> Circ. Res. 94, 866-873 (2004).</li><li>Mehta, S., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporting+from+the+field:+genetically+encoded+fluorescent+reporters+uncover+signaling+dynamics+in+living+biological+systems.">Reporting from the field: genetically encoded fluorescent reporters uncover signaling dynamics in living biological systems.</a> Annu. Rev. Biochem. 80, 375-401 (2011).</li><li>Zhang, J., Campbell, R. E., Ting, A. Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+new+fluorescent+probes+for+cell+biology.">Creating new fluorescent probes for cell biology.</a> Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).</li><li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+spatial+and+temporal+dynamics+of+intracellular+signaling.">Visualization of the spatial and temporal dynamics of intracellular signaling.</a> Dev. Cell. 4, 295-305 (2003).</li><li>Bunemann, M., Frank, M., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gi+protein+activation+in+intact+cells+involves+subunit+rearrangement+rather+than+dissociation.">Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 16077-16082 (2003).</li><li>Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+using+genetics+and+molecular+design+to+understand+complex+physiology.">Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology.</a> J. Physiol. 578, 55-67 (2007).</li><li>Willoughby, D., Cooper, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+cAMP+dynamics.">Live-cell imaging of cAMP dynamics.</a> Nat. Methods. 5, 29-36 (2008).</li><li>Nikolaev, V. O., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cAMP+synthesis+and+degradation+in+living+cells.">Monitoring of cAMP synthesis and degradation in living cells.</a> Physiology (Bethesda). 21, 86-92 (2006).</li><li>Tanimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Fluorescence+Resonance+Energy+Transfer-based+Biosensors+for+the+Quantitative+Analysis+of+Inositol+1,4,5-Trisphosphate+Dynamics+in+Calcium+Oscillations.">Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations.</a> J. Biol. Chem. 284, 8910-8917 (2009).</li><li>Nikolaev, V. O., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+for+real-time+monitoring+of+cGMP+in+living+cells.">Novel techniques for real-time monitoring of cGMP in living cells.</a> Handb. Exp. Pharmacol. , 229-243 (2009).</li><li>Nausch, L. W., Ledoux, J., Bonev, A. D., Nelson, M. T., Dostmann, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+patterning+of+cGMP+in+vascular+smooth+muscle+cells+revealed+by+single+GFP-linked+biosensors.">Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 365-370 (2008).</li><li>Borner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+measurements+of+intracellular+cAMP+concentrations+and+cAMP+analog+permeability+in+intact+cells.">FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells.</a> Nat. Protoc. 6, 427-438 (2011).</li><li>Nikolaev, V. O., Bunemann, M., Hein, L., Hannawacker, A., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+single+chain+cAMP+sensors+for+receptor-induced+signal+propagation.">Novel single chain cAMP sensors for receptor-induced signal propagation.</a> J. Biol. Chem. 279, 37215-37218 (2004).</li><li>Hong, K. P., Spitzer, N. C., Nicol, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+molecular+toolkit+for+cAMP+studies+in+live+cells.">Improved molecular toolkit for cAMP studies in live cells.</a> BMC Res. Notes. 4, 241-24 (2011).</li><li>Niino, Y., Hotta, K., Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+live+cell+imaging+using+dual+FRET+sensors+with+a+single+excitation+light.">Simultaneous live cell imaging using dual FRET sensors with a single excitation light.</a> PLoS One. 4, e6036 (2009).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat. Protoc. 1, 1057-1065 (2006).</li><li>Brumbaugh, J., Schleifenbaum, A., Stier, G., Sattler, M., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-+and+dual-parameter+FRET+kinase+probes+based+on+pleckstrin.">Single- and dual-parameter FRET kinase probes based on pleckstrin.</a> Nat. Protoc. 1, 1044-1055 (2006).</li><li>Aoki, K., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+small+GTPase+activity+with+fluorescence+resonance+energy+transfer-based+biosensors.">Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors.</a> Nat. Protoc. 4, 1623-1631 (2009).</li></ol>

אין ניגודי האינטרסים הכריזו.

בפרוטוקול זה, אנו מדגימים כיצד לבנות מערכה בעלות נמוכה אבל חזקה סריג פשוטה ליישומי הדמיה שיגרתי עם מגוון רחב של חיישנים ביולוגיים זמינים. המערכת שהוצגה כאן מיועדת לCFP וYFP, או סוגים דומים של חלבוני ניאון, כזוג-acceptor התורם. בינתיים, biosensors הבודד האחר שיהיו זמין להשתמש למשל...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב / הציוד </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> BES מאגר הכיתה </td><td> AppliChem </td><td> A1062 </td><td></td></tr><tr><td> 2 dihydrate CaCl </td><td> סיגמה אולדריץ </td><td> C5010 </td><td></td></tr><tr><td> coverslides זכוכית </td><td> Thermo Scientific </td><td> 004710781 </td><td> קוטר 24 מ&quot;מ </td></tr><tr><td> רצפת זכוכית מנות תא תרבות </td><td> מכשירי Precision העולם </td><td> FD3510-100 </td><td></td></tr><tr><td> בינוני D-MEM </td><td> Biochrom AG </td><td> F0445 </td><td></td></tr><tr><td> עוברי עגל בסרום (FCS) </td><td> Thermo Scientific </td><td> SH30073.02 </td><td></td></tr><tr><td> L-גלוטמין </td><td> Biochrom AG </td><td> K0283 </td><td></td></tr><tr><td> HEPES </td><td> סיגמא </td><td> H4034 </td><td></td></tr><tr><td> KCl </td><td> סיגמא </td><td> P5405 </td><td></td></tr><tr><td> MgCl 2 hexahydrate </td><td> AppliChem </td><td> A4425 </td><td></td></tr><tr><td> NaCl </td><td> AppliChem </td><td> A1149 </td><td></td></tr><tr><td> Na 2 HPO 4 </td><td> סיגמה אולדריץ </td><td> S9707 </td><td></td></tr><tr><td> פניצילין / סטרפטומיצין </td><td> Biochrom AG </td><td> A2213 </td><td></td></tr><tr><td> מיקרוסקופ פלואורסצנטי ההפוך </td><td> למשל ניקון </td><td> בקשה בניקון </td><td></td></tr><tr><td> CoolLED </td><td> CoolLED </td><td> p E-100 </td><td> 440 ננומטר </td></tr><tr><td> DualView </td><td> Photometrics </tד&gt; <td> Dv2-SYS </td><td></td></tr><tr><td> מחוון מסנן DualView </td><td> Photometrics </td><td> 05-EM </td><td></td></tr><tr><td> סט CFP / YFP מסנן </td><td> טכנולוגית Chroma </td><td> 49052 </td><td> ללא פליטת המסנן </td></tr><tr><td> מצלמת ORCA-03G </td><td> Hamamatsu פוטוניקס </td><td> C8484-03G02 </td><td></td></tr><tr><td> Arduino לוח קלט / פלט </td><td> Sparkfun אלקטרוניקה </td><td> Dev-00666 </td><td></td></tr><tr><td> תא התא Attofluor </td><td> Invitrogen </td><td> -7816 </td><td></td></tr><tr><td> מחשב אישי עם מערכת WindowsXP או Windows7 </td><td> כל ספק </td><td></td><td> כולל כונן קשיח עם קיבולת גבוהה </td></tr></tbody></table>

fret microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors

העברת אנרגית פורסטר תהודה (סריג) מיקרוסקופיה ממשיכה לצבור עניין גובר כטכניקה לניטור בזמן אמת של אירועים ביוכימיים ואיתות בתאים ורקמות חיות. בהשוואה לשיטות קלסיות ביוכימיים, טכנולוגיה חדשנית זו מתאפיינת ברזולוציה של זמן ומרחב גבוהה. סריג ניסויים להשתמש biosensors השונה גנטי מקודד אשר יכול לבוא לידי ביטוי, וצלם לאורך הזמן באתר או בvivo 1-2. biosensors הטיפוסי יכול לדווח על אינטראקציות בין חלבונים על ידי מדידת הסריג בין זוג fluorophore מתויג-של חלבונים או שינויים קונפורמציה בחלבון יחיד שמטפחים תורם וfluorophores acceptor מקושר עם מחצית מחייבת למולקולה של עניין 3-4. biosensors bimolecular לאינטראקציות בין חלבונים כולל, למשל, בונה נועד לפקח הפעלת G-חלבון בתאים 5, בעוד חיישני unimolecular meaשינויים קונפורמציה Suring נמצאים בשימוש נרחב לשליחים שניים תמונה כגון סידן 6, 7-8 מחנה, פוספטים אינוסיטול 9 ו cGMP 10-11. כאן אנו מתארים כיצד לבנות epifluorescence אישית סריג מערכת הדמיה ממרכיבים זמינים מסחרי בודדים ואיך לשלוט בכל ההתקנה באמצעות מנהל freeware-מייקרו. מכשיר פשוט אך רב עצמה זו נועד למדידות שגרתיות או מתוחכמות יותר סריג בתאים חיים. תמונות נרכשות מעובדות באמצעות תוספות עצמיות כתובות לדמיין שינויים ביחס סריג בזמן האמת במהלך כל ניסויים לפני שמאוחסן בתבנית גרפית תואם עם build-בfreeware ImageJ משמש לניתוח נתונים שלאחר מכן. עלות מערכת נמוכה זה מאופיין בגמישות גבוהה וניתן להשתמש בו בהצלחה לניטור אירועים ביוכימיים שונים ומולקולות איתות על ידי שפע של סריג biosensors זמין בתאים ורקמות חיות. כדוגמה, אנחנו מדגימים hאוו לשימוש במערכת הדמיה זה כדי לבצע ניטור בזמן אמת של cAMP בתאי 293A חיים על גירוי עם אגוניסט וחסמי רצפטור β-adrenergic.

Watch this Scientific Journal Video about FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors at JoVE.com

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

העברת אנרגית פורסטר תהודה (סריג) מיקרוסקופית היא טכניקה רבה עצמה לניטור בזמן אמת של אירועי איתות בתאים חיים באמצעות חיישנים ביולוגיים שונים כעיתונאים. כאן אנו מתארים כיצד לבנות epifluorescence אישית סריג מערכת הדמיה ממרכיבים מסחריים זמינים וכיצד להשתמש בו לניסויי סריג.

סריג מיקרוסקופי לניטור בזמן אמת של אירועי איתות בתאים חיים באמצעות Biosensors Unimolecular

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

fret-microscopy-for-real-time-monitoring-signaling-events-live-cells

Research

JoVE Journal

Biology

23.0K Views.  Georg August University Medical Center, Göttingen, Germany. העברת אנרגית פורסטר תהודה (סריג) מיקרוסקופית היא טכניקה רבה עצמה לניטור בזמן אמת של אירועי איתות בתאים חיים באמצעות חיישנים ביולוגיים שונים כעיתונאים. כאן אנו מתארים כיצד לבנות epifluorescence אישית סריג מערכת הדמיה ממרכיבים מסחריים זמינים וכיצד להשתמש בו לניסויי סרי...

Video: FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

שילוב QD-סריג ו מיקרופלואידיקה לפקח Nanocomplex DNA הרכבה עצמית בזמן אמת

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

ניטור בזמן אמת של אינטראקציות יגנד רצפטור עם פלואורסצנטי תהודת העברת אנרגיה

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

ניטור כפול פלסמיד בתאי יונקים חיים דרך מספר דורות על ידי מיקרוסקופ פלואורסצנטי

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

תאי יונקים האוטונומיים bioluminescent לניטור רציף וזמן אמת של Cytotoxicity

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

הדמיה בזמן אמת של יחיד תכנון הנדסי RNA תמלילים בתאים חיים באמצעות משואות bimolecular רציומטרי

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

זיהוי של ביטוי גנים בתאים תאיים חיים של Murine, אדם וחזירי מקור באמצעות חלקיקים שכותרתו פלואורסצנטי

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

G הדמית חלבון בשילוב קולטן בתיווך Chemotaxis ואירועי האיתות שלה בתאי HL60 נויטרופילים דמויים

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

זיהוי של איתות תאיים אירועים מושרה תאים קיימא על ידי אינטראקציה עם תאים שכנים מוות של תאים אפופטוטיים שעברו

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

אפיון של אירועים קלציפיקציה שימוש אופטי חיה מיקרוסקופית אלקטרונים טכניקות בתוך ימית תולעת-שפופרת

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

גירוי התמונה על ידי לייזר מפעיל בלייזר-האות-מוסדרים-קינאז (טיפשה) איתות או האירועים מיטוכונדריאלי בתאי היעד

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...