Name: isolation and intravenous injection of murine bone marrow derived monocytes
Uploaded: 2014-12-27T15:00:00.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes at JoVE.com

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.

이 연구는 동물 보호 (24D-9168.11-1 / 2008-24)에 대한 독일 법률 제 8 항에있어서, 작센 및 작센 - 안할 트, Regierungspraesidium 드레스덴 / 할레의 국가의 허가 하였다. 1 세포 분리 대퇴골과 경골의 1.1 준비 <ol><li> 폐쇄함에 이소 플루 란을 기화 5 % 이소 플루 란을 이용하여 농도 마우스를 마취. 마우스가 3 초 동안 이동을 중지 한 후, 자궁 경부 전위를 수행합니다. </li><li> 에탄올 (96 %)로 두 다리를 소독. 피부와 근육을 제거하고 멸균 메스와 뼈를 분리합니다. <ol><li> 사타구니 피부 절개로 시작하고 내과쪽으로 절개를 확장하고 낮은 송아지 주위 피부의 원형 절개를 수행합니다. 근위을 벗겨 완전히 다리의 피부를 제거합니다. </li><li> 날카로운 메스와 대퇴골에서 대퇴사 두근 근육을 분리, 전방 peroneus 모두 제거과 비복근의 근육 그룹 및 위치 및 인대를 해부하여 고관절에서 다리 관절이 어긋나 다. </li><li> 전방 시작하고 신중하게하여 관절을 disarticulating, 다리를 회전하고 인대에 병행하여 관절 주위를 진행합니다. </li></ol></li><li> 무균 세포의 준비를 보장하기 위해 90 초 최소 96 % 에탄올로 대퇴골과 경골을 씻으십시오. 에탄올 나머지 씻어 멸균 PBS로 세척 과정을 계속합니다. 참고 : 모든 후속 단계는 오염을 방지하기 위해 멸균해야합니다. </li></ol> 골수의 1.2 수확 <ol><li> 대퇴골과 경골의 축에 접근하도록 미세 가위 뼈 각각의 근위 및 원위 단부를 잘라. 이 뼈는 쉽게 깰 때문에,이 단계에주의하십시오. </li><li> 따뜻한 배지로 뼈에 플러시 (M199 + 10 % 소 태아 혈청 (FCS)을, + 1 % 페니실린 / 스트렙토 마이신). 멸균 28 G 바늘, 1 ML의 주사기, 10 ~ 15 ml의 매체 당 사이에 사용린스 뼈. </li><li> 미세 집게로 뼈를 잡고는 반투명를 꺼질 때까지 하나 하나 씻어. 조심스럽게 세포 스트레스를 최소화하기 위해 주사기 플런저의 단부에 압력을 적용한다. </li><li> 70 ㎛의 나일론 웹을 통해 골수를 여과하고 여액을 수집. 골수의 최대 양을 추출하려면, 근위 끝에서 반복 씻어. </li><li> PBS와 체를 씻어 여과 액을 수집합니다. 조심스럽게 파편과 부드러운 교반과 피펫 팅에 의해 세포 대기업을 제거. </li></ol> 1.3 재배 <ol><li> RT에서 10 분 동안 250 XG에 세포 현탁액을 원심 분리기. 뜨는을 취소하고 매체의 약 25 ml의 세포를 재현 탁. 한 번 반복합니다. </li><li> 2 차 세척 공정 후, 6 ㎖의 배지에서 세포를 재현 탁. 트리 판 블루 50 μL와 세포 용액 50 μl를 섞는다. 광학 현미경 하에서 계수 챔버에서 세포를 카운트. (C)의 수를 계산이 식을 이용하여 용액의 ELL : <img alt="식 (1)" fo:content-width="1.3in" src="/files/ftp_upload/52347/52347eq1.jpg" /></li><li> 플레이트의 저부에 영구 부착을 방지하기 위해 6 웰 초저 부착면 플레이트상에서 세포를 시드. 물론 당 최대 6 ml로 1 ㎖ 당 10 6 세포의 농도를 사용합니다. </li><li> 세포 분화를 촉진하기 위해 20 NG / ㎖의 M-CSF와 서스펜션을 보완. </li><li> 배양은 37 ℃에서 5 일간 세포를 5 % 이산화탄소 및 매일 관찰한다. </li></ol> 세포의 1.4 수확 주의 :이 단계는 얼음을 수행하여야한다. 따라서 1.4.1 또는 1.4.2 중 하나를 진행합니다. <ol><li> 이 시점에서, 그들은 초저 부착면 판하더라도 배양 플레이트에 부착 차별화 된 대 식세포를 포함한 전체 배양 수확. <ol><li> 부드러운 피펫 팅하여 전체 문화를 수확, 4 ° C PB 용액으로 분리S, 0.5 % 소 혈청 알부민 (BSA) 및 2 mM의 EDTA. 잘 모르는 경우 현미경으로 확인 - 플레이트가 부착 세포를 나머지의 명확한 때까지 반복합니다. </li></ol></li><li> 다른 방법으로, 주로 단핵 세포를 포함하는 현탁액 세포와 상층 액을 수확하여 대 식세포를 폐기합니다. <ol><li> EDTA가없는 PBS 용액 및 0.5 % BSA와 함께 비 - 부착 세포 수확. 온화하게 대 식세포를 빠지 방지하기 위해 너무 많은 힘을 가하지 마십시오. </li></ol></li></ol> 1.5 FACS 분석 (선택 사항) 참고 : MACS의 사용에 의해 CD117 + stem- 및 전구 세포의 세포 현탁액을 고갈시킬 수있다. 이 절차에 대한 제조 업체 프로토콜을 사용합니다. <ol><li> 하나가 1.4.1 또는 1.4.2 단계에 따라 세포를 수확. </li><li> 4 ° C에서 10 분 동안 250 XG에서 세포를 원심 분리기 다시이 단계를 반복합니다. </li><li> FACS (b)의 300 μL에 재현 탁 적어도 25 만 셀프로브 당 uffer. </li><li> 96 웰 플레이트 (물론 당 30 μL)에 세포 솔루션을 전송합니다. </li><li> 4 ℃에서 5 분 동안 250 XG에 세포 현탁액을 원심 분리기. </li><li> 상층 액을 제거하고 각 웰 (사용 제조 업체 희석)에 항체의 25 μl를 추가합니다. </li><li> 단계 후 세포 표현형에 대한 제조 업체 프로토콜 다음 항체 (와 세포 1.5.1-1.5.6. 단핵구 / 대 식세포의 식별을 위해 일반적으로 사용되는 마커는 CD11b, F4 / 80 (그림 3), CD115을 포함 얼룩 (그림 2), GR- 1. 참고 : 셀 솔루션의 또 다른 설명은 CD4, CD8a, 중 CD11c, CD25, CD45, CD45R, CD62L, CD80, CD86, CD145, CD117, MHCⅡ, Ly6C, Ly6G 및 CD192 같은 마커를 사용하는 것이 좋습니다. 휴대 특성은 이전 12 설명했다. </li><li> 4 ° C에서 20 분 동안 프로브를 품어. </li><li> 원심 분리기 4 ° C에서 5 분 250 XG에 프로브 및 FACS 완충액 200 μL에 재현 탁은. 반복 일두 번 단계입니다. </li><li> FACS 튜브에 프로브를 전송하고 FACS 분석 (12)을 수행합니다. </li><li> 세포 분화를 분석 일 0, 3 및 5에서 FACS 분석을 수행한다 (12). </li></ol> 2. 꼬리 정맥 주입 2.1 준비 <ol><li> 약 10 분 동안 37 ℃에서 가열 플레이트에 동물을 따뜻하게. 과열 징후를 인식 할 수 승온하면서 동물을 관찰한다. </li><li> NaCl을 150 μL에, 단계 1.1 및 1.4에 고립 된 단핵 세포를 재현 탁하고, 30G 바늘을 1 ml의 인슐린 주사기로 세포를로드합니다. 가볍게 모든 단핵 세포가 주입 보장하기 위해 주사기를로드하기 전에 솔루션을 소용돌이. 항상 열 매개 부착 및 단핵구의 활성화를 방지하기 위해 얼음에 세포를 처리합니다. </li></ol> 2.2 금지 <ol><li> 정맥 주사에​​ 대한 마우스를 선택할 때 우리에 다른 쥐를 방해하지 마십시오. </li><li> 제한 수단에 마우스를 놓습니다. 너무 많은 홍보를하지 마십시오essure는 뒷다리에주의합니다. (그림 4) 참고 : 다른 방법으로, 동물의 스트레스없는 취급을 보장하기 위해 전신 마취를 사용합니다. </li><li> 마우스가 제한 수단을 닫기 전에 호흡 공간이 있는지 확인하십시오. </li></ol> 2.3 주입 <ol><li> 소독제로 주사 부위를 소독. 마우스의 꼬리에 스프레이를 최소한 1 분간 방치 할 수 있습니다. </li><li> 혈관의 가시성을 위해, 주입 부위의 위 측면 꼬리쪽으로 약간 힘을인가하여 꼬리 정맥 혈류를 중지. </li><li> 주입하기 전에 꼬리를 90도 돌립니다. (그림 5) </li><li> 45도 각도로 주입한다. 천천히 주입 및 g 당 최대 5 μl의 해치는 동물을 피하기 위해. </li><li> 실패한 주입의 표시이다 물집이 있으면 즉시 중단하고 더 근접하게 주사를 반복합니다. </li><li> 부드러운 P를 적용하여 사출 측 지혈약 1 분 동안 ressure. </li><li> 프로 시저의 끝에서, 리 트랙터를 열고 그 장에 ​​마우스를 배치했다. </li><li> 20 분은 동물이 주입에 의해 피해를 입지되지 않아 흉골 드러 누움이 유지되도록하는 마우스를 관찰합니다. 마우스가 충분한 의식을 차릴 때까지 관찰의 시간을 연장합니다. 완전히 회복 된 후에 만​​ 다른 동물의 회사에 마우스를 돌려줍니다. </li></ol>

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The authors declare that they have no competing financial interests.

우리는 골수로부터 단핵구 뮤린 다량 격리시키는 간단하고 비용 효율적인 방법을 설명한다. 단구 수율 X10 6 5 1.4을 수득 말초 혈액을 사용하는 다른 프로토콜에 비해, 우리는 단일 공여자 마우스에서 11 × 106 ± 3 × 106 단핵구 높은 수율을 얻을 수있다. 이 방법의 문제점을 고려할 때, 비 멸균 조건 하에서 작업 할 때 오염 가능성을 언급하는 것이 중요?...

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><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&amp;nbsp;</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&amp;reg; 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&amp;uuml;lke&amp;amp;Mayr GmbH, Norderstedt, Germany</td><td></td><td>Kodan Tinktur forte</td></tr><tr><td>Disposable scalpel No.10&amp;nbsp;</td><td>Feather safety razor Co.Ltd, Osaka, Japan&amp;nbsp;</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%&amp;nbsp;</td><td>Otto Fischar GmbH und Co KG, Saarbr&amp;uuml;cken, Germany</td><td></td><td></td></tr><tr><td>Extraction unit Pipetus</td><td>Hirschmann Laborger&amp;auml;te GmbH &amp;amp; Co.KG, Eberstadt, Germany</td><td></td><td></td></tr><tr><td>F4/80</td><td>AbD Serotec, D&amp;uuml;sseldorf, Germany</td><td>MCA497APC</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td>Manufactured by our group with single components</td><td></td><td>PBS, 0.5% BSA, 0.1% NaN&lt;sub&gt;3&lt;/sub&gt;</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&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td><td></td><td></td></tr><tr><td>Falcon&amp;reg; 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&amp;ouml;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&amp;nbsp;</td><td>Labotect GmbH, G&amp;ouml;ttingen, Germany&amp;nbsp;</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&amp;szlig;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 &amp;deg;C</td></tr><tr><td>Macrophage-Colony Stimulating Factor</td><td>Sigma Aldrich, Hamburg, Germany</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/srp3110?lang=de&amp;amp;region=DE&quot;&gt;SRP3110&amp;nbsp;&lt;/a&gt;</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 &amp;deg;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&amp;nbsp;</td><td>Manufactured by our group with single components</td><td></td><td>PBS, 0.5% BSA, 2 mM 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>NaN&lt;sub&gt;3 &lt;/sub&gt;(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&amp;ouml;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 &amp;micro;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&amp;micro;l/100&amp;micro;l/200&amp;micro;l/1,000&amp;micro;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>1 ml Omnifix&lt;sup&gt;&amp;reg; &lt;/sup&gt;-F insuline syringe</td></tr><tr><td>Tubes with cap</td><td>Greiner bio one GmbH, Frickenhausen, Germany</td><td></td><td>15 ml/50 ml 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.

뮤린 골수로부터 추출 된 세포 용액은 다양한 종류의 세포로 구성된다. 주요 종류의 세포는 림프구, 과립구와 단핵구 수 있습니다. 세포 유형은 분화 5 일 후에 수확 네이티브 현탁 세포도 1에 도시되어 크기 및 입도 (granularity)에 의해 추정 될 수있다. 재배 동안 이동 셀룰러 조성합니다. 그러나 인구의 정확한 분류는 세포 마커의 독특한 표현에 의존해야합니다. M...

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).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

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

23.6K 조회수.  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).

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

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

연구

면역학 및 감염병학

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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.

Murine 골 복 막 대 식 세포 활성화에 대 한 항 체 기반 약물 효과의 평가

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

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Intravital 현미경 Monocyte 유도 및 주변 동맥 질병의 Murine 모델에서 종양 관련 신생의

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

의학

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.

NO-Redox 생물학의 연구를 위한 골수 유래 대식세포의 분리 및 시험관내 배양

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.

마우스로부터 골수 유래 수지상 세포를 분리하고 배양하기 위한 경제적이고 효율적인 프로토콜

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

신생아 골수 유래 대식세포의 식세포 분석 평가 

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.

저자 스포트라이트: Developing a Reproducible Method for Isolating Bone Marrow-Derived macrophages from Neonatal Mice to Advance Early Life Immune Response(신생아 마우스에서 골수 유래 대식세포를 분리하여 조기 면역 반응을 촉진하기 위한 재현성 있는 방

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.

신생아 마우스 골수 분리 및 골수 유래 대식세포의 준비

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

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

분리 및 설치류 골수의 정맥 주사 유래 단핵구

요약

더 많은 비디오 탐색

이 비디오의 챕터

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

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