עבודה זו מומנה על ידי תוכנית גילוי NSERC הפרט, NSERC / CIHR הבריאות שיתופי תוכנית המחקר (CHRP), ו &#39;יצירה&#39; NSERC הקנדי תוכנית הדרכה לאסטרוביולוגיה (CATP).

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+ligand-exchange+for+efficient+solubilization+of+CdSe/ZnS+quantum+dots+in+water:+A+procedure+guided+by+computational+studies.">Modified ligand-exchange for efficient solubilization of CdSe/ZnS quantum dots in water: A procedure guided by computational studies.</a> Langmuir. 24, 5270-5276 (2008).</li><li>Narayanaswamy, A., Feiner, L. F., Meijerink, A., Zaag, P. J. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+and+dot+size+on+the+spectral+properties+of+colloidal+InP/ZnS+core-shell+quantum+dots.">The effect of temperature and dot size on the spectral properties of colloidal InP/ZnS core-shell quantum dots.</a> Acs Nano. 3, 2539-2546 (2009).</li></ol>

אין ניגוד עניינים הצהיר.

חלקיקים מייצגים גישה מבטיחה יצירת אנטי מיקרוביאליים סוכנים חדשים. ניתוח עקומת הצמיחה היא דרך לעקוב אחר צפיפות תא החיידק שמבדיל תאים באופן פעיל גידול של צמיחה עכבות תאים. כאשר יחד עם ספירת צלחת, היא מאפשרת ניתוח מעמיק של הפוטנציאל של אנטיביוטיקה המצומד. פורמט 96-מאפשר ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> מספר קטלוגי </td><td> הערות (לא חובה) </td></tr><tr><td> Borate רכיב מאגר # 1 </td><td> דיג </td><td> חומצת בור, 74-1 </td><td></td></tr><tr><td> Borate רכיב מאגר # 2 </td><td> Sigma-Aldrich </td><td> נתרן Tetraborate B9876 </td><td></td></tr><tr><td> MPA </td><td> Sigma-Aldrich </td><td> M5801 </td><td></td></tr><tr><td> Vivaspin 500 </td><td> GE Healthcare </td><td> 28-9322 </td><td> MWCO השונים זמין </td></tr><tr><td> צלוחיות זכוכית </td><td> דיג </td><td> 03-338C </td><td></td></tr><tr><td> EDC </td><td> Sigma-Aldrich </td><td> E6383 </td><td></td></tr><tr><td> Polymyxin B </td><td> Sigma-Aldrich </td><td> P1004 </td><td></td></tr><tr><td> חיידקי גרםowth בינוני (LB) רכיב # 1 </td><td> דיג </td><td> NaCl S271 </td><td></td></tr><tr><td> צמיחה בינונית רכיב חיידקי (LB) # 2 </td><td> BD </td><td> Tryptone 211705 </td><td></td></tr><tr><td> בינוני התפתחות חיידקים (LB) Component # 3 </td><td> BD </td><td> תמצית שמרים 211929 </td><td></td></tr><tr><td> מנורה חשיפה לאור </td><td> מנהג </td><td></td><td></td></tr><tr><td> ברור-96-התחתונה גם צלחות </td><td> דיג </td><td> 07-200-567 07-200-730 או </td><td></td></tr><tr><td> הקרינה ספקטרומטר </td><td> התקנים מולקולריים </td><td></td><td></td></tr><tr><td> הצלחת ספיגת הקורא </td><td> התקנים מולקולריים </td><td></td><td></td></tr><tr><td> BactoAgar מדיה מוצקים </td><td> Bioshop </td><td> AGR001.1 </td><td></td></tr><tr><td> צלחות פטרי סביב </td><td> דיג </td><td> 08-75-12 </td><td></td></tr><tr><td> פטרי כלים מלבניים </td><td> דיג </td><td> 08-757-11A </td><td></td></tr></tbody></table>

solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

נקודות קוונטיות (QDs) הם חלקיקים מוליכים למחצה ניאון עם ספקטרום פליטת תלוי בגודל כי יכול להיות נרגש מתוך בחירה רחב של אורכי גל. QDs משכו הרבה עניין הדמיה, אבחון וטיפול בשל הקרינה בהיר, יציב שלהם 1,2 3,4,5. QDs יכול להיות מצומדת למגוון ביולוגי פעיל מולקולות של קשירה חיידקים ותאי יונקים 6. QDs גם נחקר באופן נרחב כסוכני ציטוטוקסיות של סיכול ממוקד של חיידקים. הופעתם של זנים עמידים להתרבות חיידקים הוא הופך במהירות למשבר בריאות הציבור, במיוחד במקרה של פתוגנים גראם שליליים 7. בשל אפקט ידוע מיקרוביאלית של ננו מסוימים, במיוחד AG, יש מאות מחקרים הבוחנים את הרעילות של חלקיקים חיידקים 8. מחקרים חיידקים בוצעו עם סוגים אחרים של חלקיקים מוליכים למחצה, כמו גם, especially טיו 2 9,10-11, אלא גם ZnO 12 ואחרים כולל CuO 13. השוואות של כמה זני חיידקים בוצעו מחקרים אלה, בדרך כלל משווים זן גראם שלילי עם גראם חיובי. עם כל החלקיקים האלה, מנגנונים של רעילות מיוחסות חמצון: או photogeneration של מינים החמצן מגיב (ROS) על ידי חלקיקים או שחרור ישיר של יונים של מתכות העלולות לגרום לרעילות חמצוני. אפילו עם חומרים אלו, תוצאות של מחקרים שונים נבדלים מאוד. בחלק מהמחקרים זן בדיקה חיובית גראם הוא מדווח רגיש יותר מ 10 גרם שלילי, במקרים אחרים זה 14 מול. מחקרים אלה נבדקו גם 15. בכל המחקרים nanoparticle, בהרכב החלקיקים, גודל, כימיה של פני השטח, מדגם הזדקנות / פירוט, ואת אורך גל, עוצמה, משך החשיפה לאור כל אלה יכולים להשפיע באופן דרמטי את התוצאות. בנוסף, synthesiתוצרי לוואי S ו ממיסים יש לקחת בחשבון 16 17. תפוקה גבוהה בטכניקות ההקרנה יש צורך להיות מסוגל לפתח יעיל nanomedicine סוכנים חדשים. QDs CdTe יש השפעות אנטי מיקרוביאליים 18 בלבד או בשילוב עם אנטיביוטיקה. במחקר קודם, הראינו כי צימוד של אנטיביוטיקה כדי CdTe יכול להגדיל רעילות לחיידקים אך להקטין רעילות לתאי יונקים, עקב ירידה בייצור של מינים החמצן מגיב מן conjugates 19. אמנם אין זה סביר כי קדמיום המכילים תרכובות תאושר לשימוש בבני אדם, הכנות כאלה עלולים לשמש חיטוי של משטחים או עיקור של מים. בפרוטוקול זה, אנחנו נותנים גישה ישירה אל solubilizing QDs CdTe עם חומצה mercaptopropionic (MPA). QDs מוכנים לשימוש בתוך שעה. לאחר מכן להפגין צימוד לסוכן מיקרוביאלית. החלק השני של פרוטוקולמדגים assay עיכוב 96-גם חיידקי באמצעות QDs מצומדות ו unconjugated. צפיפות אופטית נקרא על שעות רבות, המתיר את ההשפעות של תוספת QD וחשיפה לאור לעבור הערכה מיידית, כמו גם לאחר תקופת ההחלמה. כמו כן, אנו ממחישים ספירת המושבה לכימות הישרדות חיידקים.

Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

חלקיקים כגון מוליכים למחצה נקודות קוונטיות (QDs) ניתן להשתמש כדי ליצור סוכני photoactivatable עבור יישומים אנטי מיקרוביאליים או נגד סרטן. טכניקה זו מראה כיצד מים solubilize Telluride קדמיום (CdTe) QDs, להטות להם אנטיביוטיקה, ולבצע assay עיכוב חיידקי על בסיס עקומות גדילה ספירת צלחת.

Solubilization ביו נטיה של נקודות קוונטיות ועל מבחני רעילות של חיידקים עקומת צמיחה הרוזן פלייט

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

solubilization-bio-conjugation-quantum-dots-bacterial-toxicity-assays

Research

JoVE Journal

Bioengineering

14.2K Views.  McGill University, Montreal, QC Canada. חלקיקים כגון מוליכים למחצה נקודות קוונטיות (QDs) ניתן להשתמש כדי ליצור סוכני photoactivatable עבור יישומים אנטי מיקרוביאליים או נגד סרטן. טכניקה זו מראה כיצד מים solubilize Telluride קדמיום (CdTe) QDs, להטות להם אנטיביוטיקה, ולבצע assay עיכוב חיידקי על בסיס עקומות גדילה ספירת צלחת.

Video: Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

אקסון צמיחה מתיחה: Mechanotransduction הצמיחה העצבית

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Immobilization חיידקית עבור הדמיה על ידי מיקרוסקופית כוח אטומי

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

דור ושחזור של spheroids β-תא מהידרוג שלב צמיחת PEG-פפטיד

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

ייצור ומיקוד של חד-אנרגיה קוונטית נקודות

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

מיקרוסקופי אלקטרונים בהירי-עור גומל באמצעות חלקיקי נקודה קוונטית

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

הכנה ואפיון של נוזל HDL מחקה ננו-חלקיקים עבור אנקפסולציה גורם העצב הגדילה

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

20 mJ, 1 PS Yb: YAG דיסק דק מגבר משובי

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

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

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

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

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

זיהוי של אנדוטוקסין ניסוחים-Nano באמצעות מבחני Lysate (LAL) Amoebocyte Limulus

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

קיבעון של אנשים חיים Caenorhabditis elegans באמצעות שבב Microfluidic של דקים ועומדים בטמפרטורות Polydimethylsiloxane עם החזקת מים

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

צמיחה ואפיון של אורגנואידים לקרינה מבלוטות החלב

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...