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פרוטוקול זה הותאם מ לוץ ואח&#39; 1. קציר ועיבוד של מח העצם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בידוד של עצם השוק לבין עצם הירך: להרדים את העכבר תורם ולרסס את הרגליים עם אתנול 70%. תפוס את הקרסול first היטב עם מלקחיים בוטה מתחילים לחתוך את העור ואת שרירי הבסיסי לחשוף את עצם השוק. כדי למנוע פגיעה בעצמות, לחתוך לאט במקביל השוקה, והשאיר על כנו מפרק הברך. כדי לנקות את עצם הירך, מפרק הברך לשתק את ידי אחיזה עם מלקחיים בוטה. לנקות את השרירים עם זוג מספריים חדות מלקחיים מעוקל. המשך כלפי מעלה עד מפרק הירך חשוף ומספריים יכול להיות ממוקם בין ראש עצם הירך ואת מפרק הירך. מוציאים את העצמות על ידי חיתוך משותף בין עצם הירך לבין המותן ומקום לתוך בופר פוספט (PBS) על קרח. </li><li style=";text-align:right;direction:rtl"> הסרת מח עצם: השרירים הנותרות נקי של עצמות בעזרת מספריים מלקחיים מעוקל. לחתוך את העצם אפיפיזות כל ולאתר את חלל במרכז. בעזרת מזרק 10 מ&quot;ל עמוסה PBS ו 25G0.5 &quot;מחט, שטוף מוח העצם לתוך צלחת פטרי ללא רקמה מצופה. </li><li style=";text-align:right;direction:rtl"> עיבוד של מוח העצם: השתמש סוף הגומי של הבוכנה של מזרק 1mL לנתק את מח העצם לתוך ההשעיה תא בודד (להשתמש מעלה ומטה, לא מגרד תנועה). איסוף מח העצם בצינור בז חרוטי, ולשטוף את מוח העצם שיורית עם PBS. </li></ol> התרבות של תאים דנדריטים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאים Resuspend בוושינגטון התקשורת לספור מבשרי DC על hemocytometer (איור 1). אתה תראה תאים בגדלים שונים; מבשרי DC הם הגדולים והמבריקים. דיפרנציאלי לספור רק את התאים האלה. משתי עצמות הירך ושני tibias של C57Bl wild-type / 6 העכבר (6-8 שבועות), אתה יכול לצפות בין 25-40 x 10 6 מבשרי DC הכולל. <img src="/files/ftp_upload/old/769_2008_6_30_3/article769_figure_1.jpg" alt="" width="218" height="217" /> איור 1 </li><li style=";text-align:right;direction:rtl"> התרבות תאים בצפיפות של 2 x 10 5 מבשרי DC / mL בוושינגטון התקשורת בתוספת 40 ng / mL רקומביננטי Murine GM-CSF. פלייט תאים שאינם רקמת מצופה צלחות פטרי קלקר. </li></ol> הוסף מדיה (יום 3) כדי לרענן את התקשורת, להוסיף חצי מהנפח הכולל של התקשורת טריים בתוספת 40 ng / mL GM-CSF. החלף שליש התקשורת (יום 6) ו ההבשלה כדי לרענן את התקשורת, להסיר בזהירות שליש מהנפח הכולל של התקשורת להחליף את נפח התקשורת עם טרי DC בתוספת 40 ng / mL GM-CSF ביום 6 של התרבות. אם תרצה, תאים דנדריטים יכול להיות מגורה על התבגרות באמצעות ציטוקינים או חיוג כמו ligands הקולטן. בסרטון, DCs היו התבגר ידי גירוי לילה עם 5 ng / mL CPG. קציר של תאים דנדריטים תרבות הבירה הושלמה (איור 2). תאים יהיה גם השעיה דבק רופף הצלחת. תאים דבק ניתן להסיר על ידי גירוד מגרד את המנה עם תרבית רקמה ושטיפה עם PBS. המספר הכולל של תאים יגדל פי 5-8 במהלך התרבות שבוע ארוך תקופת הבחנה, אם כן, מצפים לקצור 1-1.6 x 10 6 תאים / מ&quot;ל. <img src="/files/ftp_upload/old/769_2008_6_30_5/article769_figure_2.jpg" alt="איור 2" width="407" height="358" /> איור 2 ריאגנטים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> DC התקשורת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> RPMI-1640 מדיה, בתוספת: </li><li style=";text-align:right;direction:rtl"> 10% בסרום שור עוברית </li><li style=";text-align:right;direction:rtl"> 100 IU / mL פניצילין </li><li style=";text-align:right;direction:rtl"> 100 מיקרוגרם / מ&quot;ל ​​סטרפטומיצין </li><li style=";text-align:right;direction:rtl"> 1% L-גלוטמין </li><li style=";text-align:right;direction:rtl"> 0.1% 2-mercaptoethanol </li><li style=";text-align:right;direction:rtl"> 1X שאינם חיוניים חומצות אמינו </li><li style=";text-align:right;direction:rtl"> 1X נתרן pyruvate </li></ol></li><li style=";text-align:right;direction:rtl"> רקומביננטי Murine GM-CSF <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> rmGM-CSF (Peprotech inc, ניו ג&#39;רזי, לא קטלוג. 315-03) </li></ol></li></ol>

<ol>
	<li>Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> J. Immunol. Methods. 223, 77-92 (1999).</li><li>Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC-based+cancer+vaccines.">DC-based cancer vaccines.</a> J. Clin. Invest. 117, 1195-1203 (2007).</li></ol>

DCs שימושיים מחקרים של אינטראקציות החיסון המולדת ובעלי כושר הסתגלות, והוא יכול להיות מועסקים וקטור חיסון. בסרטון הזה, אנחנו הוכיחו את הצעדים כדי לבודד מח עצם להבדיל תאים דנדריטים מיאלואידית במבחנה. בעקבות התקופה תרבות, תאים אלו יכולים להיות דמיינו מיקרוסקופית שני תאים כמו חסיד, ?...

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<td>recombinant murine GM-CSF</td>
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culture of myeloid dendritic cells from bone marrow precursors

תאים דנדריטים מיאלואידית (DCS) משמשים לעתים קרובות כדי לחקור את יחסי הגומלין בין מנגנוני החיסון המולדת ובעלי כושר הסתגלות לבין תגובה מוקדמת לזיהום. כי אלה האנטיגן החזק ביותר בהצגת תאים, DCs נמצאים בשימוש יותר ויותר וקטור חיסון ללמוד את האינדוקציה של אנטיגן ספציפי התגובה החיסונית. בסרטון הזה, אנחנו מדגימים את הליך קצירת tibias ואת עצמות הירך של העכבר תורם, עיבוד מח עצם הבחנה DCs במבחנה. המאפיינים של שינוי בעקבות גירוי DCs: תאים דנדריטים בשלים הם phagocytes חזק, ואילו DCs בוגרים המסוגלים מצגת אנטיגן ואינטראקציה עם CD4 + ו - CD8 + T תאים. שינוי זה בפעילות תפקודית מתכתב עם upregulation של סמנים משטח התא ייצור ציטוקינים. סוכנים רבים שניתן להשתמש בהם כדי DCs בוגרים, כולל ציטוקינים חיוג כמו ligands הקולטן. בסרטון הזה אנחנו מדגימים השוואות תזרים cytometric הביטוי של שני שיתוף גירוי מולקולות, CD86 ו - CD40, ואת ציטוקינים, IL-12, בעקבות גירוי לילה עם טיפול CPG או מדומה. לאחר התמיינות, DCs ניתן להשפיע יותר כמו וקטור חיסון לשימוש או לייצר אנטיגן ספציפי על ידי המערכת החיסונית במבחנה פועם באמצעות פפטידים או חלבונים, או transduced באמצעות וקטורים ויראליים רקומביננטי.

Watch this Scientific Journal Video about Culture of myeloid dendritic cells from bone marrow precursors at JoVE.com

Culture of myeloid dendritic cells from bone marrow precursors

וידאו זה מדגים את נוהל המבדילים תאים דנדריטים מיאלואידית ממח העצם העכבר. בידוד של השוקה העכבר הירך, ועיבוד של מח העצם הם הפגינו. תמונות הוכחת מורפולוגיה תא לפני ואחרי בידול, דמויות המתארות פנוטיפ התא IL-12 בייצור התבגרות הבאות באמצעות CPG מוצגים.

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 ...

culture-of-myeloid-dendritic-cells-from-bone-marrow-precursors

Research

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Biology

22.4K Views.  McMaster University. וידאו זה מדגים את נוהל המבדילים תאים דנדריטים מיאלואידית ממח העצם העכבר. בידוד של השוקה העכבר הירך, ועיבוד של מח העצם הם הפגינו. תמונות הוכחת מורפולוגיה תא לפני ואחרי בידול, דמויות המתארות פנוטיפ התא IL-12 בייצור התבגרות הבאות באמצעות CPG מוצגים.

Video: Culture of myeloid dendritic cells from bone marrow precursors

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

ES תאים אנושיים: תרבות החל מ תאים קפואים

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

תרבות עצבית עכבר מקדימים לתאי גזע

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

הדור של מח עצם נגזר תאים דנדריטים Murine לשימוש 2-פוטון הדמיה

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for 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 MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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 ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

התמיינות של תאים עובריים לתוך מבשרי Oligodendrocyte

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

גזירת יעיל של מבשרי לב cardiomyocytes האדם מן האדם תאים pluripotent גזע עובריים עם אינדוקציה מולקולה קטנה

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

במעקב 4 ממדי Vivo של תאי גזע ואבות היווצרות דם במבוגרי עכבר calvarial מח עצם

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

ביטוי של חלבונים פיוז'ן פלואורסצנט תאים דנדריטים הנגזרות מח עצם מאתר ו מקרופאגים

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...