عزل وحقن في الوريد من الفئران نخاع العظام المستمدة حيدات
Summary
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).
Abstract
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%± 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%± 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.
Introduction
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 ± 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.
Protocol
Representative Results
Discussion
نحن تصف طريقة بسيطة وفعالة من حيث التكلفة لعزل كميات كبيرة من وحيدات الفئران من نخاع العظام. بالمقارنة مع غيرها من البروتوكولات باستخدام الدم المحيطي، التي تحصل على عائدات الوحيدات 5 من 1.4 X10 6، ونحن قادرون على الحصول على عائدات أعلى من 11 × 10 6 ± 3 × 10 <sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center).
Thanks to Hans-Holger Gärtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.
Materials
| 6-well-ultra-low-attachment plate | Corning Incorporated, NY, USA | 6-well-ultra-low-attachment plate, with cap, sterile | |
| 8- 12 week old, male, balb/c mice | Charles River, Sulzfeld, Germany | ||
| 96-well-plate | Greiner bio one GmbH, Frickenhausen, Germany | ||
| Blue dead cell stain | Life technologies GmbH, Darmstadt, Germany | ||
| Bovine serum albumine | GE Healthcare, Freiburg, Germany | Fraction V, pH 7,0 | |
| Canules | B. Braun, Melsungen AG, Melsungen, Germany | 28G, 30G | |
| CD115 | eBioscience, San Diego, USA | 12-1152 | |
| CD11b | eBioscience, San Diego, USA | 53-0112 | |
| Cell culture dish | Greiner Bio-One GmbH, Frickenhausen, Germany | With cap, steril | |
| Centrifuge | Beckman Coulter GmbH, Krefeld, Germany | Allegra® X-15R centrifuge | |
| Depilatory cream | Veet, Mannheim, Germany | ||
| Disinfection agent | Schülke&Mayr GmbH, Norderstedt, Germany | Kodan Tinktur forte | |
| Disposable scalpel No.10 | Feather safety razor Co.Ltd, Osaka, Japan | ||
| EDTA | Sigma Aldrich, Hamburg, Germany | ||
| Ethanol 96% | Otto Fischar GmbH und Co KG, Saarbrücken, Germany | ||
| Extraction unit Pipetus | Hirschmann Laborgeräte GmbH & Co.KG, Eberstadt, Germany | ||
| F4/80 | AbD Serotec, Düsseldorf, Germany | MCA497APC | |
| FACS buffer | Manufactured by our group with single components | PBS, 0,5% BSA, 0,1% NaN3 | |
| FACS device | Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA | BD FACS Canto II | |
| FACS tubes | Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA | ||
| Falcon® pipette | Becton Dickenson Labware, NY, USA | ||
| Fetal calf serum | Sigma Aldrich, Hamburg, Germany | ||
| Fine forceps | Rubis, Stabio, Switzerland | ||
| Gloves | Rösner-Matby Meditrade GmbH, Kiefersfelden, Germany | ||
| Gr1 | eBioscience, San Diego, USA | 53-5931 | |
| Heating plate | Labotect GmbH, Göttingen, Germany | Hot Plate 062 | |
| Incubator | Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany | Incu safe | |
| Isofluran | Baxter Deutschland GmbH, Unterschleißheim, Germany | ||
| Light microscope | Carl Zeiss SMT GmbH, Oberkochen, Germany | Axiovert 40 °C | |
| Macrophage-Colony Stimulating Factor | Sigma Aldrich, Hamburg, Germany | SRP3110 | |
| Mechanical shaker | IKA, Staufen, Germany | ms2 minishaker | |
| Medium 199 | PAA Laboratories GmbH, Pasching, Austria | Warm in 37 °C water bath before use | |
| Micro test tubes | Eppendorf AG, Hamburg, Germany | ||
| Microbiological work bench | Thermo Electron, LED GmbH, Langenselbold, Germany | Hera safe | |
| Monocyte wash buffer | Manufactured by our group with single components | PBS, 0,5% BSA, 2mM EDTA | |
| Mouse restrainer | Various | ||
| NaCl | Berlin Chemie AG, Berlin, Germany | ||
| NaN3 (sodium acide) | Sigma Aldrich, Hamburg, Germany | ||
| Neubauer counting chamber | Paul Marienfeld GmbH und Co.KG, Lauda-Königshofen, Germany | ||
| Nylon cellsieve | Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA | Cell strainer, 70 µm mesh size | |
| Penicillin/Streptomycin | Sigma Aldrich, Hamburg, Germany | ||
| Phosphate buffered saline | Life technologies GmbH, Darmstadt, Germany | pH 7.4, sterile | |
| Pipettes | Eppendorf AG, Hamburg, Germany | 10µL/100µL/200µL/1000µL | |
| Pipetting heads | Eppendorf AG, Hamburg, Germany | ||
| Serological pipette | Greiner Bio-One GmbH, Frickenhausen, Germany | Cellstar 5 ml, 10 ml | |
| Suction unit | Integra bioscience, Fernwald, Germany | Vacusafe comfort | |
| Surgical scissors | Word Precision Instruments, Inc., Sarasota, USA | ||
| Syringe | B. Braun, Melsungen AG, Melsungen, Germany | 1mL Omnifix® -F insuline syringe | |
| Tubes with cap | Greiner bio one GmbH, Frickenhausen, Germany | 15mL/50mL Cellstar tubes | |
| Warm water bath | Julabo Labortechnik GmbH, Seelbach, Germany | Julabo SW22 |
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