עבודה זו מומנה על ידי המכון הקנדי לבריאות מחקר (CIHR) (גרנט MOP-119,503) ואת למדעי הטבע וההנדסה המועצה של קנדה (NSERC). NSERC גם נתמך studentships הקיץ י&quot;ד ו- AA י&quot;ד נתמך על ידי אוניברסיטת קולומביה הבריטית (UBC) עם פרס המילגה 4 שנים, AA נתמך על ידי CIHR עם פרס שני סטודנט לתואר שני (CGS-M). אנו מודים קלווין Roskelley לסיוע עם מיקרוסקופ השתמשו כדי ליצור את התמונות באיור 2. כמו כן, אנו מכירים תמיכה מן מתחם Cytometry Animal ושפל UBC.

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The authors have no financial conflicts of interest.

בכתב היד הזה, אנו מספקים שיטה להפקת מקרופאגים GM-CSF נגזר DCs מתרבה מח עצם עכבר אחת כי הוא עיבוד לוץ et al. 6. ביטוי MHCII ו FL-HA מחייב מבחינה בין DCs בשלה מקרופאגים בתרבות זו (ראו איור 3 ג), אשר בעבר כבר קשה. זה, יחד עם דו&quot;ח נוסף על ידי Helft et al. 19, הדגים הט...

כמה בשיטות תרבות במבחנה תוארו ליצור מקרופאגים עצם שמקורם במח (BMDMs) ו DCs עצם שמקורם במח (BMDCs) באמצעות אחד או שילוב של גורמי גדילה. BMDMs יכול להיווצר על ידי culturing תאים במח העצם או באמצעות גורם מגרה המושבה מקרופאג (M-CSF) או GM-CSF 1,2. לקבלת BMDCs, תוספת של ליגנד FLT3 לתרבות מח עצם מוליד DCs קלאסית plasmacytoid לא חסיד (CD11c גבוה / גבוהה CD11c MHCII lo, B220 + בהתאמה) לאחר 9 ימים בתרבות 3,4. לעומת זאת, תאים שאינם חסידים שנוצרו לאחר 7 עד 10 ימים בתרבות עם GM-CSF לבד 5,6, GM-CSF ו- IL-4 7, או GM-CSF ו FLT3 ליגנד 8,9 ליצור BMDCs יותר מקרוב דומת DCs הדלקתית (גבוה CD11c, MHCII גבוהה CD11b +) 10. בעוד תרבויות חוץ גופית אלה משמשים ליצירת מקרופאגים אוDCS, לא ברור אם כל תרבות מולידה אוכלוסיות טהורות. לדוגמא, אם כי תאים חסידים בתרבויות-CSF GM מתוארים להיות מקרופאגים 5, התאים לא חסידים מאותה התרבות משמשים DCs 6,11-13, עם ההנחה כי הם הומוגנית וכל השתנות שנצפתה היא עקב שלבי ההתפתחות השונים 14,15. יתר על כן, מחקרים מצאו GM-CSF כדי להוות גורם צמיחה חיוני להתפתחות מקרופאג המכתשית in vivo 16,17, וניתן להשתמש במבחנה כדי לייצר מקרופאגים מכתשיים דמוי 16,17,18. מלבד דבקות, הנהלים להפקה מאקרופאגים DCs ממח עצם מטופלים-CSF GM תרבויות דומים מאוד המצביע על ההטרוגניות יכול להתקיים בתוך תרבויות מח עצם GM-CSF. זה אכן נראה את המקרה כפי שני מסמכים לדווח על נוכחות של BMDMs בשבריר לא חסיד של תרבויות BMDC. במאמר אחד, הם identified אוכלוסייה של תאים כמו CD11c +, CD11b +, באמצע MHCII, MerTK +, ו CD115 +, אשר הביע חתימה ביטוי גנים שדמה יותר מכול מקרופאגים מכתשיים והיה לו יכולת מופחתת להפעיל תאי T 19. המאמר השני בשימוש MHCII ו FL-HA לזהות אוכלוסייה המכתשית מקרופאג דמוי (+ CD11c, MHCII אמצע / נמוך, FL-HA גבוהה) כי היה נבדל לא מפותחים (+ CD11c, MHCII באמצע, FL-HA נמוך) ובוגרת DCS (CD11c +, MHCII גבוהה), הן phenotypically והן מבחינה תפקודית 18. שני העיתונים הללו ממחישים כי GM-CSF תרבויות BMDC הן הטרוגניים, המכילים את שני מקרופאג ואוכלוסיות DC המציין טיפול שיש לנקוט כאשר לפרש נתונים מתרבויות BMDC. פרוטוקול זה מתאר כיצד לבודד מח עצם, תאי מח עצם התרבות GM-CSF, ולזהות את מכתשי מקרופאג דמויהאוכלוסייה מן DCs בשלה ובוגרת בתרבות מח העצם על ידי cytometry זרימה באמצעות FL-HA מחייב וביטוי MHCII. הליך זה מבוסס על הליך הוקמה של לוץ ואח 6 והוא מסוגל לייצר 5 -. 10 x 10 6 תאים לא חסיד ביום 7 של תרבות 10 מ&quot;ל. התרבות היא שמיש מימים 7 עד 10 ומניבת אוכלוסייה הטרוגנית של מקרופאגים, DCS בשלה ובוגרת, כמו גם אבות חלקים ביום 7. זה מספק שיטה פשוטה לגדול ולבודד במבחנה המכתשית דמוי מקרופאגים בכמויות גדולות.

<table><tbody><tr><td>Flow Cytometer</td><td>BD&amp;nbsp;</td><td>LSR-II</td><td></td></tr><tr><td>Automated Inverted Microscope&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>DMI4000 B</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Thermo Fisher</td><td>ST-40R</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Nuaire</td><td>NU-425-600</td><td></td></tr><tr><td>Syringe 1 ml</td><td>BD</td><td>309659</td><td></td></tr><tr><td>26 1/2 Gauge Needle</td><td>BD</td><td>305111</td><td></td></tr><tr><td>50 ml Conical Tube&amp;nbsp;</td><td>Corning</td><td>357070</td><td>*Falcon brand</td></tr><tr><td>Eppendorf tubes (1.5 ml)</td><td>Corning</td><td>MCT-150-C</td><td></td></tr><tr><td>5 ml polystyrene round bottomed tubes</td><td>Corning</td><td>352052</td><td></td></tr><tr><td>Dissection Tools</td><td>Fine Science Tools&amp;nbsp;</td><td>*Various&amp;nbsp;</td><td>*Dissection scissors, dumont forcep and standard forcep&amp;nbsp;</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Reichert</td><td>1490</td><td></td></tr><tr><td>Sterile 100 mm x 15 mm Petri Dish</td><td>Corning</td><td>351029</td><td>*Falcon brand</td></tr><tr><td>2-Mercaptoethanol</td><td>Thermo Fisher</td><td>21985-023</td><td></td></tr><tr><td>Ammonium Chloride</td><td>BDH</td><td>BDH0208-500G</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Bioreagents</td><td>BP1600-1</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651-5MG</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134-1KG</td><td>Ethylenediaminetetraacetic acid</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</td><td>16000-044</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermo Fisher</td><td>14175-095&amp;nbsp;</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>15630-080</td><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G8540-100G</td><td></td></tr><tr><td>LPS Ultrapure</td><td>Invivogen</td><td>tlrl-3pelps</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution&amp;nbsp;</td><td>Thermo Fisher</td><td>11140-050</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>BDH</td><td>BDH0268-500G</td><td></td></tr><tr><td>Potassium Chloride</td><td>BDH</td><td>BDH9258-500G</td><td></td></tr><tr><td>Recombinant GM-CSF</td><td>Peprotech</td><td>315-03-A</td><td></td></tr><tr><td>Rooster Comb Sodium Hyaluronate&amp;nbsp;</td><td>Sigma</td><td>H5388-1G</td><td>*Used to make fluoresceinated hyaluronan</td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Thermo Fisher</td><td>21870-076</td><td>No sodium pyruvate no glutamine. Warm media to 37&amp;nbsp;&amp;#176;C before using.</td></tr><tr><td>Sodium Chloride</td><td>Fisher&amp;nbsp;</td><td>5271-10&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma</td><td>50876-1Kg</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P5290-100G</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Fisher Bioreagents</td><td>BP152-5</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant</td><td>N/A</td><td>N/A</td><td>Clone: 2.4G2</td></tr><tr><td>Anti-CD11c PeCy7</td><td>eBioscience</td><td>25-0114-82</td><td>Clone: N418</td></tr><tr><td>Anti-Gr-1 efluor450</td><td>eBioscience</td><td>48-5931-82</td><td>Clone: RB6-8C5</td></tr><tr><td>Anti-MHCII APC</td><td>eBioscience</td><td>17-5321-82</td><td>Clone: M5/114.15.2</td></tr><tr><td>Biotinylated Anti-MerTK</td><td>Abcam</td><td>BAF591</td><td>Goat polyclonal IgG</td></tr><tr><td>Streptavidin PE</td><td>eBioscience</td><td>12-4317-87</td><td></td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4170-25MG</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Sigma</td><td>D9542-5MG</td><td></td></tr></tbody></table>

generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow

המקרופאגים ותאים דנדריטיים (DCS) הם תאים חיסוניים מולדים נמצא ברקמות ואיברים הלימפה כי לשחק תפקיד מפתח בהגנה מפני פתוגנים. עם זאת, הם קשים לבודד בכמות מספקת כדי ללמוד אותם בפירוט, ולכן, במבחנת מודלים פותחו. בתרבויות במבחנה של מקרופאגים עצם שמקורם במח ותאי דנדריטיים הם ומבוססים ושיטות בעל ערך עבור מחקרים אימונולוגית. כאן, שיטת culturing וזיהוי הוא DCS ו מקרופאגים מתרבה אחת של תאים במח עצם עכבר העיקרי באמצעות גורם מגרת מושבת מקרופאג גרנולוציט ציטוקינים (GM-CSF) מתוארת. פרוטוקול זה מבוסס על ההליך הוקם ראשון שפותח על ידי ואח &#39;לוץ. בשנת 1999 עבור DCs עצם שמקורם במח. התרבות היא הטרוגנית, הכוללת MHCII ו hyaluronan fluoresceinated (FL-HA) משמשים כדי להבחין מקרופאגים DCs בשלה ובוגרת. GM-CSF אלה מתקבלים מקרופאגים פרוvide מקור נוח של מקרופאגים נגזר במבחנה כי הדומים מקרופאגים מכתשיים בשני הפנוטיפ ותפקוד.

תרשים זרימה המסכם את השלבים העיקריים של שיטה זו מוצגת באיור 1. הצפיפות והמורפולוגיה של תרבות מח עצם בזמנים שונים של התרבות מוצגות באיור 2. באותו יום 1, התאים הם קטנים ודלילים אבל ביום 3, יש יותר תאים, וחלקם גדולים יותר ועוד כמה החלו לד?...

Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

דור וזיהוי GM-CSF נגזר מכתשיים דמוי מאקרופאגים דנדריטים תאי עכבר מח עצם

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

generation-identification-gm-csf-derived-alveolar-like-macrophages

Research

JoVE Journal

Immunology and Infection

17.9K Views.  University of British Columbia. Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages. 

Video: Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

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 ...

Biology

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

חקירה של קיטוב Macrophage שימוש המקרופאגים נגזרים מח עצם

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

אפיון חילוף חומרים של מקוטב M1 והמקרופאגים נגזר מח עצם M2 באמצעות ניתוח תאי שטף בזמן אמת

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

דור מספר גדול של תאים מיאלואידים אבות ו מבשרי תא דנדריטי ממח העצם מאתר באמצעות תא הרומן מיון אסטרטגיה

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

זיהוי ובידוד של Oligopotent, ביצע לשושלת אבות מיאלואידית ממח העצם העכבר

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

פרוטוקול חסכוני ויעיל לבידוד וגידול תאים דנדריטיים שמקורם במח העצם מעכברים

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

בידוד ותרבית של מקרופאגים שמקורם במח עצם מעכברים

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

הערכת בדיקה פגוציטית של מקרופאגים הנגזרים ממח עצם בילוד 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

זרקור על המחבר: פיתוח שיטה ניתנת לשחזור לבידוד מקרופאגים שמקורם במח עצם מעכברים ילודים לקידום תגובות חיסוניות בגיל צעיר

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

עכבר יילודים בידוד מח עצם והכנת מקרופאגים שמקורם במח עצם

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

דור וזיהוי GM-CSF נגזר מכתשיים דמוי מאקרופאגים דנדריטים תאי עכבר מח עצם

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Chapters in this video

התרבות של תאים דנדריטים מיאלואידית מ מבשרי מח עצם

חקירה של קיטוב Macrophage שימוש המקרופאגים נגזרים מח עצם

מח עצם נגזרות Macrophage הפקה

אפיון חילוף חומרים של מקוטב M1 והמקרופאגים נגזר מח עצם M2 באמצעות ניתוח תאי שטף בזמן אמת

דור מספר גדול של תאים מיאלואידים אבות ו מבשרי תא דנדריטי ממח העצם מאתר באמצעות תא הרומן מיון אסטרטגיה

זיהוי ובידוד של Oligopotent, ביצע לשושלת אבות מיאלואידית ממח העצם העכבר

פרוטוקול חסכוני ויעיל לבידוד וגידול תאים דנדריטיים שמקורם במח העצם מעכברים

בידוד ותרבית של מקרופאגים שמקורם במח עצם מעכברים

עכבר יילודים בידוד מח עצם והכנת מקרופאגים שמקורם במח עצם

עכבר יילודים בידוד מח עצם והכנת מקרופאגים שמקורם במח עצם