המחברים מבקשים להודות מתקן מיקרוסקופיה אופטית מתקדמת (www.aomf.ca) בבית רשת בריאות באוניברסיטת לסיוע עם מיקרוסקופיה, ומר ג&#39;ייסון אליס מן חנות הנסיכה מרגרט Cancer Center מכונות להכנת הב&quot;ש והשלב הדמיה. אנו רוצים גם להודות לד&quot;ר איריס Kulbatski לעריכת כתב יד.

<ol>
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יש המחברים שום אינטרסים כלכליים מתחרים.

הדמיה בזמן אמת, סידורי של התהליכים התאיים הדינמיים במח עצם מספקת מידע כי הוא מאתגר אחרת להשיג באמצעות טכניקות קונבנציונליות כגון היסטולוגיה וספירת דם מוחלטת. מודל WC הירך המתואר כאן מספק הזדמנויות ייחודיות לחקור שינויים הסלולר מבניים במח העצם לאורך זמן. למרות מודל WC ...

מוח עצם הוא איבר חשוב המעורב hematopoiesis ורגולציה חיסונית. הוא מורכב מרכיב hematopoietic המכיל תאי גזע אבות היווצרות דם (HSPCs), ומרכיב סטרומה המכיל ובתאים הלא hematopoietic כי להצמיח תאי mesenchymal 1. שני שלישים של פעילות hematopoietic מוקדש לדור של תאים מיאלואידית 2. בפרט, מספר רב של נויטרופילים מיוצר במח העצם, עם 1-2 x 10 11 תאים שנוצרו ליום בתוך אדם מבוגר נורמלי 2. נויטרופילים הם קו ההגנה הראשון מפני זיהומים מיקרוביאליים שמורות בעיקר במח העצם עד מתח מפעילה התגייסותם להשלים נויטרופילים היקפי 1,3. בנוסף להשפעות שלהם אנטי מיקרוביאליים, מחקרים אחרונים מראים תפקיד חשוב של נויטרופילים בביולוגיה סרטן, נתקל בשני פרו פנוטיפים אנטי tumorigenic תלוי צמיחה הפיכתבטא גורם (TGF-β) איתות 4,5 microenvironment הגידול. יתר על כן, מחקרים הראו כי נויטרופילים שמצטברים גידולים ראשוניים להשפעות פרו-tumorigenic ו גרורתי ידי דיכוי הפונקציה ציטוטוקסיות של תאי T 6,7, תוך נויטרופילים במחזור להפעיל ציטוטוקסיות, אנטי-גרורתי השפעה 8. ככזה, החקירה של תאי hematopoietic במח העצם, במיוחד נויטרופילים, היא חיונית כדי הבהרת תפקידם בתקנה חיסונית גידול. Histopathology וספירת דם היקפית מלאה משמשים באופן שיגרתי כדי להעריך שינויים הסלולר מבניים מח עצם 9. עם זאת, שיטות אלה רק לספק מידע סטטי של אוכלוסיות תאים שונות או microstructures רקמות. אורך in vivo הדמיה ניתן להשתמש בשילוב עם השיטות הסטנדרטיות כדי להעריך את הדינמיקה של מרכיבים תאיים, כלי דם ואת סטרומה מרובים וכן תא אל גאינטראקציות ell באופן אורכי. מיקרוסקופיה Intravital (IVM), מוגדר הדמיה של חיים חיים ברזולוציה מיקרוסקופית 10, הוא שימושי במיוחד להערכת תהליכים תאיים דינמיים לאורך זמן באותו המדגם, צמצום מספר בעלי חי ניסוי הנדרש. IVM היא לעתים קרובות בשילוב עם תא חלון מושתלים כרוני (WC) לגשת איבר עניין הדמיה במשך זמן של שבועות עד חודשים. מודלי WC גולגולתי ו-skinfold הגבו יש את ההיסטוריה הארוכה ביותר של שימוש שראשיתה אמצע 1990. לאחרונה, מודלים WC איבר ספציפי אחרים כגון אלה של כרית שומן החלב ואת אברי הבטן השונים פותחו 11. הגישה הטיפוסית הדמית מח עצם in vivo יש בעיקר מעורבת חשיפת מִכסֵה הַגוּלגוֹלֶת של עכברים, שם העצם הדליל מאפשר הדמיה ישירה של תאים בודדים עם התערבות כירורגית מינימאלית 12-14. עם זאת, מח עצם calvarial עשוי bדואר שונה מזו של עצמות אחרות, כגון עצם הארוך, כפי שהוכח על ידי מספר נמוך יותר של HSPCs ותאי היפוקסי ב מִכסֵה הַגוּלגוֹלֶת, מעידה מופחתת תחזוקה ופיתוח של HSPCs 15. לכן, גישות חלופיות להערכת מרכיבים תאיים ב העצם הארוך נחקרו. אלה כוללים חשיפה ישירה של מח עצם ירך 16 והשתלות אקטופי של ירך פילוג skinfold הגבה בב&quot;ש 17. עם זאת, לשעבר הוא הליך מסוף שאינו מאפשר מעקב של שינויים הסלולר, מבניים ותפקודיים לאורך תקופות זמן ארוכות, ואת הסיכוי האחרון מפריע פונקצית מח עצם נורמלית עקב השתלת עצם הירך לאתר אקטופי בתוך WC הגבה skinfold. שיטה נוספת המאפשרת הדמיה סדרתי orthotopic של מח עצם הירך לאורך זמן הוא השימוש של WC בעצם הירך. בדוח הקודם אחת הפגינו הדמיה לטווח ארוך מיקרו מחזורי במח העצם הירך באמצעותירך WC בעכברים 18. בנוסף, החוקרים הדגימו ויזואליזציה של תאים סרטניים בעצם הירך, המציין השירות שלה גרור במח עצם ניטור. עם זאת, עיצוב בב&quot;ש זה היה מוגבל על ידי גודל גדול שלה (בקוטר 1.2 ס&quot;מ) ואזור הדמיה קטן יחסית (4 מ&quot;מ קוטר), אשר היה מתאים רק עכברים גדולים (26-34 גרם, 3-6 חודשים של גיל) ובכך להפוך את להתקרב מעשי לשימוש שגרתי. לכן, אסלה חדשה עם גודל כולל קטן ואזור הדמיה הפנימית גדול נועדו לצורך מחקר זה. מטרת המחקר הנוכחי הייתה לקבוע שיטת הדמית סוגי תאים שונים במח עצם הירך. מודל WC הירך פותח בתוך הבית ושמש לדמיין ולעקוב אחר נויטרופילים בתוך רשת כלי דם 3D. באמצעות מודל זה, IVM של מח העצם ניתן לבצע באופן סדרתי על 40 ימים. גישה זו ניתן ליישם במגוון תחומי הבהרת תהליכי hematopoiesis, תקנה חיסוניתהתפתחות גידולים nd.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NRCNU-F athymic nude mice</td>
			<td>Taconic</td>
			<td>Ncr nude</td>
			<td>8-10 weeks old, female</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>JB1302P</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Bioniche Animal Health Canada, Inc.&#160;</td>
			<td>DIN 01989529</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer HealthCare, Bayer Inc.</td>
			<td>DIN 02169592</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Proxima</td>
			<td>DYNJP2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric heating pad</td>
			<td>Life Brand</td>
			<td>57800827375</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (tear gel)</td>
			<td>Novartis&#160;</td>
			<td>T296/2</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% betadine</td>
			<td>Purdue Frederick Co</td>
			<td>67618-151-16</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol</td>
			<td>GreenField</td>
			<td>P010IP7P</td>
			<td></td>
		</tr>
		<tr>
			<td>10% betadine</td>
			<td>Purdue Frederick Co</td>
			<td>67618-150-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle (#3)</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade (#15)</td>
			<td>VWR</td>
			<td>89176-368</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors curved</td>
			<td>Fine science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Baby-Mixter Hemostat</td>
			<td>Fine science Tools</td>
			<td>13013-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>Fine science Tools</td>
			<td>11151-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostats</td>
			<td>Fine science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill</td>
			<td>Harvard Apparaus</td>
			<td>72-6065</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill burrs</td>
			<td>Fine Science Tools</td>
			<td>19007-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Femur window chamber</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>9.1mm- 8.5mm- 7.5 mm (outer to inner diameter), 2.16 mm (radius of two holes), 13.9mm (distance between two holes), 0.7mm (thickness)</td>
		</tr>
		<tr>
			<td>U-shaped bar</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>13.8mm (length), 1.6 mm (width), 3.7mm (height)</td>
		</tr>
		<tr>
			<td>Coverglass (8mm)</td>
			<td>Warner Instruments&#160;</td>
			<td>HBIO 64-0701 CS-8R</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring (8mm)</td>
			<td>ACKLANDS GRAINGER</td>
			<td>UNSPSC # 31163202</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuts (hexagon stainless steel)</td>
			<td>Fastenal</td>
			<td>70701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>3M</td>
			<td>RelyX U200</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (5-0 Monosof black)</td>
			<td>Covioien</td>
			<td>SN-5698</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder</td>
			<td>Fine Science Tools</td>
			<td>12501-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Reckitt Benckiser</td>
			<td>DIN 0281251</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim</td>
			<td>DIN 02240463</td>
			<td></td>
		</tr>
		<tr>
			<td>Amoxicillin (Clamavox)</td>
			<td>Pfizer</td>
			<td>DIN 02027879</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD2000S</td>
			<td></td>
		</tr>
		<tr>
			<td>APC- Anti-Mouse Ly-6G (Gr-1)&#160;</td>
			<td>eBioscience</td>
			<td>17-9668</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscope LSM 710</td>
			<td>Carl Zeiss</td>
			<td>Zeiss LSM 710 NLO</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging stage</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>15.9cm (length), 11cm (width), 0,9cm (height)</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>Imaris 8.0</td>
			<td>Image analysis software described in Section 3 of the Protocol&#160;</td>
		</tr>
		<tr>
			<td>Zen 2012</td>
			<td>Zeiss</td>
			<td>Zen 2012</td>
			<td>Image acqusition software described in Section 2 of the Protocol&#160;</td>
		</tr>
	</tbody>
</table>

femur window chamber model for in vivo cell tracking in the murine bone marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

מח עצם ירך Murine הוא לגשת בהצלחה באמצעות WC לאפשר ויזואליזציה של נויטרופילים פרט רשתות כלי דם. איור 1 מציג את מכשיר WC ומתאר את ההליך כירורגי, אשר כרוך בחשיפה של העצם ודילול הירך של עצם קורטיקלית כדי לקבל גישה אופטית בתוך עצם. הניתוח ונסבל היטב ע?...

Watch this Scientific Journal Video about Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow at JoVE.com

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

חלון ירך דגם קאמרי<em&gt; In vivo</em&gt; מעקב תא Murine מח עצם

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

femur-window-chamber-model-for-vivo-cell-tracking-murine-bone

Research

JoVE Journal

Immunology and Infection

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

8.1K Views.  Princess Margaret Cancer Centre. The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Video: Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

ב גיבוש biofilm חוץ גופית ב 8-Slide גם לשכת

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

הדור וסימון של Murine מח עצם הנגזרות תאים דנדריטים עם nanocrystals Qdot ללימודי מעקב

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

אינדוקציה של מחלת שתל נגד מארח ו<em&gt; In Vivo</em&gt; ניטור תא T תוך שימוש במודל Murine MHC התאמה

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

מח עצם נגזרות Macrophage הפקה

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

המונוציטים בידוד והזרקה תוך ורידית של Murine מח העצם נגזר

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

מעקב עכבר מח עצם המונוציטים<em&gt; In vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Murine הינד לימב ארוך עצם Dissection ובידוד מח העצם

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

מיון תא מופעל קרינה עבור טיהור של תאי דנדריטים Plasmacytoid ממח עצם העכבר

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

זיהוי של Inflammasome ההפעלה ומוות תא Pyroptotic של מקרופאגים הנגזרות מח עצם מאתר

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

לטווח ארוך In Vivo מעקב של תאים דלקתיים הדינמיקה בתוך דרוזופילה הגלמים

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

חלון ירך דגם קאמרי

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