קציר מוח העצם מן עצמות הירך לבין Tibiae <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השלב הראשון של ההליך הוא לקצור מח העצם של עצמות הירך לבין tibiae של 6 - עכבר 12 שבועות הישן (אנחנו משתמשים BALB / c עכברים אבל אתה יכול לעשות הליך זה עם זן כל העכבר). כדי להתחיל, להרדים את העכבר משאיפת CO 2 (לא יוצג). קציר עצם הירך לבין tibiae מן הרגליים האחוריות של כל אחד. ואז, עם סכין גילוח, לחתוך כל סוף עצמות הירך כדי להסיר את הירך והמפרקים הברך לחשוף את מח. </li><li style=";text-align:right;direction:rtl"> באופן דומה, לחתוך כל סוף tibiae כדי להסיר את הברך ואזורים סמוכים הקרסול לחשוף את מח. </li><li style=";text-align:right;direction:rtl"> קח מזרק 3 מ&quot;ל עם מחט 26-מד ולמלא אותו RPMI FBS בתוספת 10%, פניצילין, סטרפטומיצין, ואת בטא mercaptoethanol (BME 100 = UM). בעזרת פינצטה, להחזיק את העצם מעל צלחת פטרי המכילה RPMI התקשורת. הכנס את מחט המזרק לתוך קצהו האחד של העצם, לוחץ על המתג כדי לשטוף את מוח העצם. מחט המזרק ניתן להזיז למעלה ולמטה בתוך העצם כדי לשטוף את מוח שיורית. </li><li style=";text-align:right;direction:rtl"> חזור על התהליך עבור כל העצמות, ואז להמשיך עם הקמת תרבויות. </li></ol> הקמת תרבויות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להקים את התרבויות מח עצם, פיפטה מח העצם סמוקות מעלה ומטה מספר פעמים כדי לשבור את הרקמה לתוך תא ההשעיה. </li><li style=";text-align:right;direction:rtl"> מקום מסנן 70 מיקרון על גבי צינור חרוטי 50 מ&quot;ל, ומוסיפים את ההשעיה תא המסנן. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את צלחת פטרי פעם אחת עם RPMI, ואז מוסיפים את לשטוף את מסנן 70 מיקרון. </li><li style=";text-align:right;direction:rtl"> שימוש בסוף גומי של 1 מ&quot;ל במזרק הבוכנה, לטחון את חתיכות מוח העצם שנותרו על גבי פילטר 70 מיקרון. </li><li style=";text-align:right;direction:rtl"> שטפו את המסנן פעם אחת עם RPMI. </li><li style=";text-align:right;direction:rtl"> לאחר שטיפת מסנן, צנטריפוגה ההשעיה תא בסל&quot;ד 1200 למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> שטפו את תא גלולה ידי resuspending ב PBS. </li><li style=";text-align:right;direction:rtl"> ספירת התאים באמצעות hemocytometer. אנחנו בדרך כלל להשיג ~ 90 מיליון תאים מהעצמות אחד העכבר (שתי עצמות הירך בתוספת שתי tibiae). לאחר ספירת תאים, צנטריפוגה אותם שוב בסל&quot;ד 1200 למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים כמות קטנה של RPMI ו aliquot אותם לתוך צלוחיות תרבות רקמה המכילה מספיק RPMI כך הריכוז הסופי הוא 1 מיליון תאים לכל מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוסף ציטוקינים כדי לקדם את הפיתוח של סוג התא העניין שלך. במקרה זה, ציטוקינים interleukin-3 (IL-3 = 10 ng / mL) נוסף כדי לקדם את הפיתוח של בזופילים ותאי פיטום. כדי לרכוש בזופילים, דגירה 10 ימים. עבור תאים התורן, דגירה של 5 שבועות. בעת יצירת תא הפיטום, התקשורת IL-3 יש לשנות פעם בשבוע. </li><li style=";text-align:right;direction:rtl"> הנה מבט על כמה תאים התורן אמור להופיע רק לאחר ציפוי. תמונה זו ממחישה את התמיינות של תאים במח התורן לתוך בזופילים ותאי תורן 10 ימים לאחר תוספת של interleukin-3. </li><li style=";text-align:right;direction:rtl"> פעם את סוג התא הרצוי מתקבל, להמשיך לשלב electroporation. </li></ol> Electroporating תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להתחיל את הצעד electroporation, לקבוע את מספר התאים בקבוק התרבות. </li><li style=";text-align:right;direction:rtl"> תאים הם electroporated בדרך כלל צפיפות של 10 מיליון דולר מ&quot;ל, כדי להעביר את המספר הסלולרי נדרש צינורות חרוטי ו צנטריפוגות צינורות ב 1200 סל&quot;ד במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> לאחר centrifuging, לשאוב את התקשורת, לשטוף את התאים עם 1X PBS, ו צנטריפוגות שוב ב 1200 סל&quot;ד במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> לשאוב PBS ולהוסיף Bio-Rad ג&#39;ין חיץ Pulser electroporation לעשות השעיה תא של 10 מיליון תאים לכל מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> לאחר resuspending את התאים, להוסיף את ה-DNA פלסמיד הרצוי בריכוז סופי של 10 עד 20 מיקרוגרם למ&quot;ל. </li><li style=";text-align:right;direction:rtl"> Aliquot 150 מיקרוליטר של השעיה התא לתוך בארות על פי בחירתך על צלחת electroporation 96-היטב. </li><li style=";text-align:right;direction:rtl"> שים את צלחת electroporation בחדר צלחת MXcell ולסגור את המכסה. </li><li style=";text-align:right;direction:rtl"> לפני transfection של תא הפיטום, פרוטוקול electroporation חייב להיות שתוכנת MXcell אלא באמצעות פרוטוקול מראש או מאוחסנים. באמצעות ניסויים אופטימיזציה גילינו את היעילות הגבוהה ביותר transfection של תא הפיטום להתרחש באמצעות מרובע פרוטוקולים גל הדופק. אנו ישתנו התנאים electroporation על הצלחת להעביר 300V/20ms, 350/15ms, 350V/20ms ו 350V/10ms פולסים מרובע לנופף 2000uF אוהם ו 1000. לאחר פרוטוקול הוגדר ולטעון המכשיר, ללחוץ על &quot;דופק&quot; כדי electroporate התאים. </li><li style=";text-align:right;direction:rtl"> לאחר electroporation תושלם, העברת תאים לצלחת בתרבית רקמה. אנחנו בדרך כלל העברת כל 150 electroporation מדגם microliter אל באר צלחת 48-היטב בתרבות רקמה המכילה 300 מיקרוליטר RPMI (בתוספת 10% FCS, פניצילין, סטרפטומיצין, ואת בטא mercaptoethanol) עם 10 ננוגרם למ&quot;ל interleukin-3. תאים מודגרת לילה בשעה 37 ° C, אז assayed 24 שעות מאוחר יותר לביטוי ודיכוי. </li></ol> טראןsfection תוצאות תמונת מיקרוסקופ פלואורסצנטי של תאים לאחר electroporation מוצלח באמצעות 20 מיקרוגרם למ&quot;ל GFP פלסמיד מוצג הווידאו. שימוש במערכת electroporation MXcell transfection יעילות של כ -30% ניתן להשיג. המערכת מאפשרת לך להשתנות התנאים כדי למקסם את היעילות transfection שלך, תוך שמירה על כדאיות התא.

כאשר מידע גנטי יותר עולה וכלים חדשים כגון siRNA מפותחים, השימוש פיזיולוגית התאים הרלוונטיים הפך חשוב יותר מתמיד כדי לקדם את ההבנה שלנו של המסלולים המחלה, חלבונים אינטראקציות, ואת הולכת אותות. תאים ראשוניים מתקבלים ישירות לרקמות או נוזלים מעובד במבחנה. תאים אלה ניתן לטפל במספר דרכים, כולל באמצעות הקדמה של חומר גנטי חיצוני. זה הופך ברור יותר ויותר כי הדרך היעילה ביותר להציג את ה-DNA או פלסמיד siRNA לתוך תאים ראשוניים הוא electroporation. Electroporation חושף תאים פולסים חשמליים על מנת transiently להגדיל את החדירות של קרום התא, ובכך לאפשר חומצות גרעין אקסוגניים להיכנס לתא. שיטה זו של transfection יכול להיות מותאם כדי להכיל את ההבדלים בין תאים מסוגים שונים / קווי, ולכן ניתן להשתמש בו כדי transfect תא הפיטום וכן כל מח עצם אחרות הנגזרות התא. יתר על כן, בניגוד transfection ויראלי בתיווך, electroporation אינה מטילה מגבלות על גודל של ה-DNA transfected, ולא דורשים הכנה נרחבת. בניגוד transfection השומנים בתיווך, electroporation יכול להיות פחות רעיל ואינו לגרום השמנה endosomal של חומצת גרעין transfected. ביו רד פיתחה את מערכת electroporation MXcell במיוחד עבור תאים אלה. MXcell מאפשר לך להשתנות התנאים כדי למקסם את היעילות ואת הכדאיות transfection התא. כדי לייעל את נצילות transfection ולמזער מוות של תאים, מספר רב של פרמטרים electroporation כולל מתח, קיבול, התנגדות, אורך הפולס יכול להיות מותאם ומוערכת.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gene Pulser MXcell Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2670</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2677</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation

זה הופך ברור יותר ויותר כי electroporation היא הדרך היעילה ביותר להציג את ה-DNA או פלסמיד siRNA לתוך תאים ראשוניים. Pulser ג&#39;ין מערכת MXcell electroporation וג&#39;ין Pulser חיץ electroporation פותחו במיוחד כדי transfect חומצות גרעין לתאי יונקים קשות transfect תאים, כגון וידאו cells.This יסודי גזע מדגים כיצד להקים ראשוני בתרביות תאים hematopoietic ממח העצם Murine ולאחר מכן להכין אותם electroporation במערכת MXcell. אנחנו מתחילים ידי בידוד עצם הירך לבין השוקה. מוח העצם של הירך והן השוקה נקצרים אז ותרבויות מבוססים. בתרבית תאים במח העצם הם transfected כך וניתח.

Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

הליך זה מתאר כיצד להקים ראשוני בתרביות תאים hematopoietic ממח העצם Murine ואחריו transfection באמצעות Pulser ג&#39;ין מערכת MXCell electroporation.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

<a class="grey_button" href="/video/1047">
ChineseVersion
</a>

the-preparation-primary-hematopoietic-cell-cultures-from-murine-bone

Google

Research

JoVE Journal

Biology

25.6K Views.  Bio-Rad Laboratories, Inc.  ChineseVersion 

Video: The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

גזירת בתאי גזע hematopoietic מ Murine בתאי גזע עובריים

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

תרבויות עצבית ראשונית של המוח של Late Stage תסיסנית גלמים

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporation של mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

התרבות של תאים דנדריטים מיאלואידית מ מבשרי מח עצם

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

ראשי ניתק דופמין במוח התיכון Cell תרבויות מן ילודים מכרסמים

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

ביות של תאים hematopoietic אל מח העצם

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

תרבות ראשיים electroporation פלסמיד של האיבר Murine של קורטי.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

תא תרבויות ראשי מ תסיסנית Gastrula עוברים

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

הכנת תא / תרבויות והיצמדות למנגנון הראשי myogenic מבשר משושלות חוליות בסל

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

גזירה האוסטאוקלסטים מעכבר מח עצם

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...