This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center). 
Thanks to Hans-Holger G&#228;rtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.

מחקר זה בוצע באישורה של המדינה סקסוניה וסקסוניה-אנהלט, Regierungspraesidium דרזדן / האלי, על פי סעיף 8 לחוק הגרמני להגנה על בעלי חיים (24D-9,168.11-1 / 2008-24). בידוד תא 1 1.1 הכנת עצם הירך לבין השוקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר באמצעות 5% ריכוז isoflurane על ידי אידוי isoflurane לפח סגור. לאחר העכבר הפסיק לזוז במשך 3 שניות, לבצע הנקע בצוואר הרחם. </li><li style=";text-align:right;direction:rtl"> לחטא את הגפיים האחורי עם אתנול (96%). להסיר את העור ושרירים ולהפריד את העצמות עם אזמל סטרילי. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> התחל עם חתך בעור מפשעה, ולהרחיב את החתך לכיוון malleolus המדיאלי ולבצע נתיחה חוזר של העור סביב העגל הנמוך. הסר את העור של הרגליים לחלוטין על ידי לקלף אותה proximally. </li><li style=";text-align:right;direction:rtl"> לנתק את השריר הארבע ראשי מעצם הירך עם אזמל חד, תסיר את שני peroneus הקדמיוקבוצות השרירים הגסטרוקנמיוס וdisarticulate הרגל במפרק הירך על ידי איתור ולנתח את הרצועות. </li><li style=";text-align:right;direction:rtl"> התחל anteriorly ולהמשיך סביב המפרק על ידי הסיבוב בזהירות את הרגל וtransecting הרצועות, ובכך disarticulating המשותף. </li></ol></li><li style=";text-align:right;direction:rtl"> לשטוף עצם ירך ועצם שוק עם 96% אתנול למשך תקופה מינימאלית של 90 שניות כדי להבטיח הכנת תא ספטית. להמשיך את תהליך השטיפה עם PBS סטרילי כדי לשטוף נותר אתנול. הערה: כל הצעדים הבאים חייבים להיות סטרילי כדי למנוע זיהום. </li></ol> 1.2 קצירה של מח העצם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותך את הקצה הפרוקסימלי והדיסטלי של כל עצם עם זוג מספריים קנס כדי לקבל גישה לפירי הירך ושוקה. תיזהר עם צעד זה, שכן העצמות האלה לשבור בקלות רבה. </li><li style=";text-align:right;direction:rtl"> לשטוף את העצמות עם מדיום חם (M199 10% בסרום + עגל עוברי (FCS), + 1% פניצילין / סטרפטומיצין). השתמש במחט סטרילית 28-G, מזרק 1 מיליליטר, ובין 10-15 מיליליטר בינונייםעצם לשטיפה. </li><li style=";text-align:right;direction:rtl"> החזק את העצם עם מלקחיים עדינים ולשטוף אחד אחד עד שהוא הופך שקוף למחצה. זהירות להחיל לחץ בסופו של בוכנת המזרק על מנת למזער מתח תא. </li><li style=";text-align:right;direction:rtl"> סנן את מח העצם באמצעות אינטרנט ניילון 70 מיקרומטר ולאסוף התסנין. כדי לחלץ את הכמות המרבית של מח עצם, לשטוף שוב ושוב מהקצוות דיסטלי ופרוקסימלי. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את המסננת עם PBS ולאסוף התסנין. לסלק פסולת בזהירות וקונגלומרטים סלולריים על ידי ערבוב וpipetting עדינים. </li></ol> 1.3 טיפוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה ההשעיה התא ב 250 XG במשך 10 דקות ב RT. בטל supernatant ו resuspend התאים עם כ 25 מיליליטר של מדיום. חזור פעם אחת. </li><li style=";text-align:right;direction:rtl"> Resuspend תאי 6 מיליליטר של מדיום לאחר שלב הכביסה השני. מערבבים 50 μl של פתרון התא עם 50 μl של trypan כחול. ספירת התאים בתא ספירה תחת מיקרוסקופ אור. לחשב את מספר גאמות בפתרון באמצעות נוסחה זו: <img alt="משוואת 1" fo:content-width="1.3in" src="/files/ftp_upload/52347/52347eq1.jpg" /></li><li style=";text-align:right;direction:rtl"> זרעי התאים על צלחות משטח נמוך במיוחד מצורף 6-גם למניעת הדבקה קבועה לתחתית הצלחת. השתמש בריכוז של 10 6 תאים למ&quot;ל עם עד 6 מיליליטר לכל טוב. </li><li style=";text-align:right;direction:rtl"> להשלים את ההשעיה עם M-CSF מיליליטר / 20 ng לקדם התמיינות תאים. </li><li style=";text-align:right;direction:rtl"> תרבות התאים במשך 5 ימים ב 37 מעלות צלזיוס, 5% פחמן דו חמצני ולבחון מדי יום. </li></ol> 1.4 קצירה של תאים הערה: עליך לבצע פעולות אלה על קרח. המשך עם או 1.4.1 או 1.4.2 בהתאם. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בשלב זה, לקצור את התרבות כולה, כולל מקרופאגים המובחנים שידבקו לתרבות-הצלחות, גם אם הם צלחות משטח נמוך במיוחד קובץ מצורף. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לקצור את כל התרבות על ידי pipetting העדין וניתוק עם פתרון של 4 מעלות צלזיוס PBS, 0.5% אלבומין בסרום שור (BSA) ו- 2 mM EDTA. חזור על פעולה עד הצלחות ברורות של שנותר תאים חסיד - לאשר תחת מיקרוסקופ אם אינך בטוח. </li></ol></li><li style=";text-align:right;direction:rtl"> לחלופין, לבטל מקרופאגים חסיד על ידי איסוף supernatant עם התאים בתרחיף, שבעיקר מכילים מונוציטים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קציר התאים שאינם חסיד עם פתרון של PBS ללא EDTA וBSA 0.5%. אין ליישם בכוח רב מדי על מנת להימנע מפירוק מקרופאגים המעטה חסיד. </li></ol></li></ol> 1.5 FACS-ניתוח (אופציונאלי) הערה: ניתן לרוקן את ההשעיה התא של תאי stem- ואב CD117 + על ידי השימוש בMACS. להשתמש בפרוטוקולי יצרן להליך זה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קציר התאים על פי שני שלבים 1.4.1 או 1.4.2. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה התאים ב 250 XG במשך 10 דקות ב 4 ° C ולחזור על פעולה זו שוב. </li><li style=";text-align:right;direction:rtl"> Resuspend לפחות 250,000 תאים ב300 μl של FACS בuffer לבדיקה. </li><li style=";text-align:right;direction:rtl"> העבר את פתרון תא על 96-היטב צלחת (30 μl לכל היטב). </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ההשעיה התא ב 250 XG במשך 5 דקות ב 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את supernatant ולהוסיף 25 μl של נוגדן לכל טוב (דילול יצרן שימוש). </li><li style=";text-align:right;direction:rtl"> כתם התאים עם נוגדנים (להלן פרוטוקולי יצרן לתא phenotyping לאחר צעדי 1.5.1-1.5.6. סמנים בשימוש נפוץ לזיהוי מונוציטים / מקרופאג כוללים CD11b, F4 / 80 (איור 3), CD115 (איור 2), וGr- 1. הערה: לתיאור נוסף של פתרונות סלולריים, השימוש בסמנים כמו CD4, CD8a, CD11c, CD25, CD45, CD45R, CD62L, CD80, CD86, CD145, CD117, MHCⅡ, Ly6C, Ly6G, וCD192 מומלץ. מאפיינים סלולריים תוארו בעבר 12. </li><li style=";text-align:right;direction:rtl"> דגירה הבדיקות במשך 20 דקות על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה הבדיקות ב 250 XG במשך 5 דקות על 4 מעלות צלזיוס וגלולה עם 200 μl של חיץ FACS. ה חזורהוא צעד פעמיים. </li><li style=";text-align:right;direction:rtl"> העבר את הבדיקות לתוך צינורות FACS ולבצע ניתוח FACS 12. </li><li style=";text-align:right;direction:rtl"> ביצוע ניתוח FACS 12 ביום 0, 3 ו -5 לנתח התמיינות תאים. </li></ol> הזרקה לוריד 2. זנב 2.1 הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחמם את בעלי החיים על צלחת חימום על 37 מעלות צלזיוס למשך כ -10 דקות. שים לב לבעלי החיים בזמן ההתחממות לזהות סימנים של התחממות יתר. </li><li style=";text-align:right;direction:rtl"> Resuspend מונוציטים, מבודדים בצעדי 1.1- 1.4, ב -150 μl של NaCl, ולטעון את התאים לתוך מזרק אינסולין 1 מיליליטר עם מחט 30G. קל מערבולת הפתרון לפני טעינת המזרק על מנת להבטיח את כל מונוציטים מוזרקים. תמיד להתמודד עם תאים על קרח, כדי למנוע עיקול בתיווך חום והפעלה של מונוציטים. </li></ol> 2.2 הרחקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הימנע מלהפריע עכברים אחרים בכלוב בעת בחירת עכבר להזרקה תוך ורידית. </li><li style=";text-align:right;direction:rtl"> מניחים את העכבר לעוצר. הימנע יותר מדי יחסי ציבורחסימת חצוצרות ולהיות זהירות עם הגפיים האחורי. (איור 4) הערה: לחלופין, להשתמש בהרדמה כללית כדי להבטיח טיפול ללא מתח של בעלי החיים. </li><li style=";text-align:right;direction:rtl"> ודא שיש לו את העכבר מרחב לנשימה לפני סגירת העוצר. </li></ol> 2.3 הזרקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחטא את אזור ההזרקה עם סוכן חיטוי. תרסיס על הזנב של העכבר ולתת לעמוד במשך דקות לפחות 1. </li><li style=";text-align:right;direction:rtl"> לעצור את זרימת דם ורידית של הזנב על ידי הפעלת לחץ עדין לצד זנב לרוחב מעל אתר ההזרקה, ראות טובה יותר של הוורידים. </li><li style=";text-align:right;direction:rtl"> הפעל את הזנב 90 מעלות, לפני ההזרקה. (איור 5) </li><li style=";text-align:right;direction:rtl"> להזריק בזווית של 45 מעלות. להזריק לאט ולא יותר מ 5 μl לז, כדי למנוע פגיעה בבעלי חיים. </li><li style=";text-align:right;direction:rtl"> אם יש שלפוחית, וזה סימן של הזרקה נכשלה, להפסיק מייד ולחזור על ההזרקה יותר proximally. </li><li style=";text-align:right;direction:rtl"> לעצור את הדימום בצד ההזרקה על ידי יישום p העדיןressure כ 1 דק &#39;. </li><li style=";text-align:right;direction:rtl"> בסופו של ההליך, לפתוח את מפשק ומניח את העכבר בכלוב שלה. </li><li style=";text-align:right;direction:rtl"> שים לב לעכבר במשך 20 דקות על מנת להבטיח כי בעל החיים לא נפגעו על ידי ההזרקה ומתוחזק כיבה sternal. הארך את הזמן של התבוננות עד העכבר להכרה מספקת. החזר את העכבר לחברה של בעלי חיים אחרים רק לאחר שהתאושש באופן מלא. </li></ol>

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The authors declare that they have no competing financial interests.

אנו מתארים שיטה פשוטה וחסכונית לבודד את הכמויות גדולות של מונוציטים עכבריים ממח עצם. בהשוואה לפרוטוקולים אחרים באמצעות דם היקפי, הלהשיג תשואות מונוציטים 5 של 1.4 x10 6, אנו מסוגלים להשיג תשואות גבוהות יותר של 11 x 10 6 ± 3 x 10 6 מונוציטים מעכבר תורם יחיד...

The isolation of monocytes is important and critical for many in vitro and in vivo studies. These cells are targets for diseases such as peripheral arterial disease, coronary heart disease, or other ischemic diseases, since collateral vessel growth is strongly driven by local inflammation. Inflammatory responses include endothelial activation and local recruitment of leukocytes, mainly monocytes, which then mature to macrophages and create a highly arteriogenic environment by secreting multiple growth factors to induce the remodeling of an arteriole into a functional collateral artery1-3. Monocytes also mature to dendritic cells, which are frequently used for immunological studies4,5 and cancer research6,7.
Problematic in the approach for monocyte isolation from peripheral blood8 is the high number of donor animals needed to produce a sufficient amount of monocytes for most analyses. Former protocols describe methods such as density gradient centrifugation and cell depletion via MACS9 when isolating monocytes; however, these techniques can alter the characteristics and functionality of monocytes which can lead to difficulties in interpretation10,11. Moreover, these methods are difficult and can reduce experimental reproducibility.
Our aim with this protocol is to provide a simple and cost effective method to generate large amounts of bone marrow-derived monocytes. Due to the high cell yield of 11 x 106 &#177; 3 x 106 cells obtained by this protocol, we can substantially reduce the number of mice required during the isolation of bone marrow-derived monocytes. The procedure can be completed within a minimal amount of time, and without using expensive and complicated techniques as referenced above. Here, we extract monocytes from native bone marrow suspension of donor mice, cultivate the suspension on ultralow attachment plates, and supplement the solution with 20 ng/ml M-CFS. On day 5 of incubation, cells are harvested and characterized to confirm functional and phenotypic properties.
For experiments in the field of arteriogenesis, intravenous transplantation of these bone marrow-derived monocytes into mice is an effective method of systemic drug delivery, which can be combined with femoral artery ligation in common peripheral arterial disease models.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>6-well-ultra-low-attachment plate</td>
			<td>Corning Incorporated, NY, USA</td>
			<td></td>
			<td>6-well-ultra-low-attachment plate, with cap, sterile</td>
		</tr>
		<tr>
			<td>8- 12 week old, male, balb/c mice&#160;</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Greiner bio one GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue dead cell stain</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine</td>
			<td>GE Healthcare, Freiburg, Germany</td>
			<td></td>
			<td>Fraction V, pH 7,0</td>
		</tr>
		<tr>
			<td>Canules</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td>28G, 30G</td>
		</tr>
		<tr>
			<td>CD115</td>
			<td>eBioscience, San Diego, USA</td>
			<td>12-1152</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>eBioscience, San Diego, USA</td>
			<td>53-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>With cap, steril</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter GmbH, Krefeld, Germany</td>
			<td></td>
			<td>Allegra&#174; X-15R centrifuge</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet, Mannheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection agent</td>
			<td>Sch&#252;lke&#38;Mayr GmbH, Norderstedt, Germany</td>
			<td></td>
			<td>Kodan Tinktur forte</td>
		</tr>
		<tr>
			<td>Disposable scalpel No.10&#160;</td>
			<td>Feather safety razor Co.Ltd, Osaka, Japan&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%&#160;</td>
			<td>Otto Fischar GmbH und Co KG, Saarbr&#252;cken, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extraction unit Pipetus</td>
			<td>Hirschmann Laborger&#228;te GmbH &#38; Co.KG, Eberstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>AbD Serotec, D&#252;sseldorf, Germany</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>Manufactured by our group with single components</td>
			<td></td>
			<td>PBS, 0,5% BSA, 0,1% NaN3</td>
		</tr>
		<tr>
			<td>FACS device</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td>BD FACS Canto II</td>
		</tr>
		<tr>
			<td>FACS tubes&#160;&#160;&#160;&#160;</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon&#174; pipette</td>
			<td>Becton Dickenson Labware, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Rubis, Stabio, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>R&#246;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gr1</td>
			<td>eBioscience, San Diego, USA</td>
			<td>53-5931</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate&#160;</td>
			<td>Labotect GmbH, G&#246;ttingen, Germany&#160;</td>
			<td></td>
			<td>Hot Plate 062</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td>
			<td></td>
			<td>Incu safe</td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Baxter Deutschland GmbH, Unterschlei&#223;heim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td>
			<td></td>
			<td>Axiovert 40 &#176;C</td>
		</tr>
		<tr>
			<td>Macrophage-Colony Stimulating Factor</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/srp3110?lang=de&amp;region=DE">SRP3110&#160;</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical shaker</td>
			<td>IKA, Staufen, Germany</td>
			<td></td>
			<td>ms2 minishaker</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>PAA Laboratories GmbH, Pasching, Austria</td>
			<td></td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Micro test tubes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological work bench</td>
			<td>Thermo Electron, LED GmbH, Langenselbold, Germany</td>
			<td></td>
			<td>Hera safe</td>
		</tr>
		<tr>
			<td>Monocyte wash buffer&#160;</td>
			<td>Manufactured by our group with single components</td>
			<td></td>
			<td>PBS, 0,5% BSA, 2mM EDTA</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Berlin Chemie AG, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3 (sodium acide)</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Paul Marienfeld GmbH und Co.KG, Lauda-K&#246;nigshofen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cellsieve</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td>Cell strainer, 70 &#181;m mesh size</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td></td>
			<td>pH 7.4, sterile</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td>10&#181;L/100&#181;L/200&#181;L/1000&#181;L</td>
		</tr>
		<tr>
			<td>Pipetting heads</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>Cellstar 5 ml, 10 ml</td>
		</tr>
		<tr>
			<td>Suction unit</td>
			<td>Integra bioscience, Fernwald, Germany</td>
			<td></td>
			<td>Vacusafe comfort</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td>1mL Omnifix&#174; -F insuline syringe</td>
		</tr>
		<tr>
			<td>Tubes with cap</td>
			<td>Greiner bio one GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>15mL/50mL Cellstar tubes</td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td>Julabo Labortechnik GmbH, Seelbach, Germany</td>
			<td></td>
			<td>Julabo SW22</td>
		</tr>
	</tbody>
</table>

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.

פתרון התא מופק ממוח העצם העכברי מורכב מסוגי תאים שונים. הסוגים העיקריים של התאים לימפוציטים הם, גרנולוציטים ומונוציטים. ניתן להעריך סוגי תאים על ידי גודל וגרעיניות, המוצג באיור 1 עבור שתי השעיות היליד והתאים שנקטפו לאחר 5 ימים של בידול. שים לב להרכב הסלולרי ה?...

Watch this Scientific Journal Video about Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes at JoVE.com

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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 ...

isolation-intravenous-injection-murine-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

23.3K Views.  Otto von Guericke University Magdeburg. 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).

Video: Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma וול gondii ציסטה כינונה ב הופעל מח עצם הנגזרות המקרופאגים והגבלות Bradyzoite

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

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 ...

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 ...

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, ...

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

בידוד של לינה קינאין תאים מונמוננקה מקולון מורלין באמצעות קולגנאז

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

בידוד ותרבית במבחנה של מקרופאגים שמקורם במח עצם לחקר הביולוגיה של NO-Redox

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

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 ...