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>

<ol>
	<li>Herold, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+monocytes:+a+novel+strategy+for+in+vivo+augmentation+of+collateral+vessel+growth.">Transplantation of monocytes: a novel strategy for in vivo augmentation of collateral vessel growth.</a> Hum Gene Ther. 15 (1), 1-12 (2004).</li><li>Herold, J., Tillmanns, H., Xing, Z., Strasser, R. H., Braun-Dullaeus, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transduction+of+monocytes:+promising+vehicles+for+therapeutic+arteriogenesis.">Isolation and transduction of monocytes: promising vehicles for therapeutic arteriogenesis.</a> Langenbecks Arch Surg. 391 (2), 72-82 (2006).</li><li>Francke, A., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C., Herold, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+bone+marrow+derived+monocytes:+a+novel+approach+for+augmentation+of+arteriogenesis+in+a+murine+model+of+femoral+artery+ligation.">Transplantation of bone marrow derived monocytes: a novel approach for augmentation of arteriogenesis in a murine model of femoral artery ligation.</a> American Journal Of Translational Research. 5 (2), 155-169 (2013).</li><li>Leon, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+differentiation+potential+of+mouse+monocytes:+monocytes+represent+immediate+precursors+of+CD8-+and+CD8++splenic+dendritic+cells.">Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8- and CD8+ splenic dendritic cells.</a> Blood. 103 (7), 2668-2676 (2004).</li><li>Timmerman, J. M., Levy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+vaccines+for+cancer+immunotherapy.">Dendritic cell vaccines for cancer immunotherapy.</a> Annu Rev Med. 50, 507-529 (1999).</li><li>Salem, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+dendritic+cells+for+Peptide-based+vaccination+in+cancer+immunotherapy.">The use of dendritic cells for Peptide-based vaccination in cancer immunotherapy.</a> Methods Mol Biol. 1139, 479-503 (2014).</li><li>Timmerman, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiotype-pulsed+dendritic+cell+vaccination+for+B-cell+lymphoma:+clinical+and+immune+responses+in+35+patients.">Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients.</a> Blood. 99 (5), 1517-1526 (2002).</li><li>Berthold, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+monocytes+by+Ficoll+density+gradient+centrifugation.">Isolation of human monocytes by Ficoll density gradient centrifugation.</a> Blut. 43 (6), 367-371 (1981).</li><li>Houthuys, E., Movahedi, K., De Baetselier, P., Van Ginderachter, J. A., Brouckaert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+and+purification+of+mouse+peripheral+blood+monocytes.">A method for the isolation and purification of mouse peripheral blood monocytes.</a> Journal Of Immunological Methods. 359 (1-2), 1-10 (2010).</li><li>Seeger, F. H., Tonn, T., Krzossok, N., Zeiher, A. M., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+isolation+procedures+matter:+a+comparison+of+different+isolation+protocols+of+bone+marrow+mononuclear+cells+used+for+cell+therapy+in+patients+with+acute+myocardial+infarction.">Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction.</a> European Heart Journal. 28 (6), 766-772 (2007).</li><li>Auffray, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1++CD115++CD135++common+macrophage/DC+precursors+and+the+role+of+CX3CR1+in+their+response+to+inflammation.">CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation.</a> The Journal Of Experimental Medicine. 206 (3), 595-606 (2009).</li><li>Francke, A., Herold, J., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mature+murine+monocytes+from+heterogeneous+bone+marrow+and+description+of+their+properties.">Generation of mature murine monocytes from heterogeneous bone marrow and description of their properties.</a> J. Histochem. Cytochem. 59 (9), 813-825 (2011).</li><li>Sneider, E. B., Nowicki, P. T., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+in+the+treatment+of+peripheral+arterial+disease.">Regenerative medicine in the treatment of peripheral arterial disease.</a> Journal Of Cellular Biochemistry. 108 (4), 753-761 (2009).</li><li>Van Royen, N., Piek, J. J., Schaper, W., Fulton, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+clinical+arteriogenesis+research.">A critical review of clinical arteriogenesis research.</a> J Am Coll Cardiol. 55 (1), 17-25 (2009).</li><li>Lawall, H., Bramlage, P., Amann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+peripheral+arterial+disease+using+stem+and+progenitor+cell+therapy.">Treatment of peripheral arterial disease using stem and progenitor cell therapy.</a> J Vasc Surg. 53 (2), 445-453 (2011).</li><li>Herold, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetanus+toxoid-pulsed+monocyte+vaccination+for+augmentation+of+collateral+vessel+growth.">Tetanus toxoid-pulsed monocyte vaccination for augmentation of collateral vessel growth.</a> Journal of the American Heart Association. 3 (2), e000611 (2014).</li></ol>

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

23.5K 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

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 ...

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 ...

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, ...

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

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

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

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

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

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

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

הערכה של נוגדן מבוססי תרופות השפעות על מח העצם מאתר והפעלה מקרופאג הצפק

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

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 ...

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 ...

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 ...