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.

This study was performed with permission of the State of Saxony and Saxony-Anhalt, Regierungspraesidium Dresden/Halle, according to Section 8 of the German Law for animal protection (24D-9168.11-1/2008-24).
1 Cell Isolation
1.1 Preparation of Femur and Tibia
<ol>
	<li>Anesthetize the mouse using a 5% isoflurane concentration by vaporizing isoflurane in a closed bin. After the mouse has stopped moving for 3 sec, perform the cervical dislocation.</li>
	<li>Disinfect the hind limbs with ethanol (96%). Remove skin and muscles and separate the bones with a sterile scalpel.
	<ol>
		<li>Start with an inguinal skin incision, and extend the incision toward the medial malleolus and perform a circular dissection of the skin around the lower calf. Remove the skin of the legs completely by peeling it proximally.</li>
		<li>Detach the quadriceps muscle from the femur with a sharp scalpel, remove both the anterior peroneus and the gastrocnemius muscle groups and disarticulate the leg at the hip joint by locating and dissecting the ligaments.</li>
		<li>Start anteriorly and proceed around the joint by carefully rotating the leg and transecting the ligaments, thereby disarticulating the joint.</li>
	</ol>
	</li>
	<li>Wash femur and tibia with 96% ethanol for a minimum of 90 sec to guarantee aseptic cell preparation. Continue the washing process with sterile PBS to rinse off remaining ethanol. 
	Note: All subsequent steps must be sterile to avoid contamination.</li>
</ol>
1.2 Harvesting of Bone Marrow
<ol>
	<li>Cut the proximal and distal end of each bone with a pair of fine scissors to gain access to the femoral and tibial shafts. Be careful with this step, since these bones break very easily.</li>
	<li>Flush the bones with warm medium (M199 + 10% fetal calf serum (FCS), + 1% Penicillin/Streptomycin). Use a sterile 28-G needle, a 1 ml syringe, and between 10-15 ml medium per bone for rinsing.</li>
	<li>Hold the bone with a fine forceps and rinse one by one until it turns semi-translucent. Carefully apply pressure to the end of the syringe plunger in order to minimize cell stress.</li>
	<li>Filter the bone marrow through a 70 &#181;m nylon web and gather the filtrate. To extract the maximum quantity of bone marrow, rinse repeatedly from the distal and proximal ends.</li>
	<li>Rinse the sieve with PBS and gather the filtrate. Carefully dislodge debris and cellular conglomerates by gentle stirring and pipetting.</li>
</ol>
1.3 Cultivation
<ol>
	<li>Centrifuge the cell suspension at 250 x g for 10 min at RT. Discard the supernatant and resuspend the cells with approximately 25 ml of medium. Repeat once.</li>
	<li>Resuspend the cells in 6 ml of medium after the second washing step. Mix 50 &#181;l of the cell solution with 50 &#181;l of trypan blue. Count the cells in a counting chamber under a light microscope. Calculate the number of cells in the solution using this formula: 
	<img alt="Equation 1" fo:content-width="1.3in" src="/files/ftp_upload/52347/52347eq1.jpg" /></li>
	<li>Seed the cells on 6-well ultra-low-attachment surface plates to prevent permanent adhesion to the bottom of the plate. Use a concentration of 106 cells per ml with up to 6 ml per well.</li>
	<li>Supplement the suspension with 20 ng/ml M-CSF to promote cell differentiation.</li>
	<li>Culture the cells for 5 days at 37 &#176;C and 5% carbon dioxide and observe daily.</li>
</ol>
1.4 Harvesting of Cells
Note: These steps should be performed on ice. Proceed with either 1.4.1 or 1.4.2 accordingly.
<ol>
	<li>At this point, harvest the entire culture, including differentiated macrophages that will adhere to the culture-plates, even if they are ultra-low-attachment surface plates.
	<ol>
		<li>Harvest the entire culture by gentle pipetting and detaching with a solution of 4 &#176;C PBS, 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Repeat until the plates are clear of remaining adherent cells - confirm under a microscope if unsure.</li>
	</ol>
	</li>
	<li>Alternatively, discard adherent macrophages by harvesting the supernatant with the cells in suspension, which predominantly contain monocytes.
	<ol>
		<li>Harvest the non-adherent cells with a solution of EDTA-free PBS and 0.5% BSA. Do not apply too much force in order to avoid dislodging mildly adherent macrophages.</li>
	</ol>
	</li>
</ol>
1.5 FACS-Analysis (Optional)
Note: It is possible to deplete the cell suspension of CD117+ stem- and progenitor cells by the use of MACS. Use manufacturer protocols for this procedure.
<ol>
	<li>Harvest the cells according to either steps 1.4.1 or 1.4.2.</li>
	<li>Centrifuge the cells at 250 x g for 10 min at 4 &#176;C and repeat this step again.</li>
	<li>Resuspend at least 250,000 cells in 300 &#181;l of FACS buffer per probe.</li>
	<li>Transfer cell solution onto a 96-well-plate (30 &#181;l per well).</li>
	<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and add 25 &#181;l of antibody to each well (use manufacturer dilution).</li>
	<li>Stain the cells with antibodies (following manufacturer protocols for cell phenotyping after steps 1.5.1-1.5.6. Commonly used markers for monocyte / macrophage identification include CD11b, F4/80 (Figure 3), CD115 (Figure 2), and Gr-1. 
	Note: For a further description of cell solutions, the use of markers like CD4, CD8a, CD11c, CD25, CD45, CD45R, CD62L, CD80, CD86, CD145, CD117, MHC&#8545;, Ly6C, Ly6G, and CD192 is recommended. Cellular characteristics were previously described12.</li>
	<li>Incubate the probes for 20 min at 4 &#176;C.</li>
	<li>Centrifuge the probes at 250 x g for 5 min at 4 &#176;C and resuspend with 200 &#181;l of FACS buffer. Repeat this step twice.</li>
	<li>Transfer the probes into FACS tubes and perform the FACS analysis12.</li>
	<li>Perform FACS analysis12 on day 0, 3 and 5 to analyze cell differentiation.</li>
</ol>
2. Tail Vein Injection
2.1 Preparation
<ol>
	<li>Warm the animals on a heating plate at 37 &#176;C for about 10 min. Observe the animals while warming to recognize signs of overheating.</li>
	<li>Resuspend the monocytes, isolated in steps 1.1- 1.4, in 150 &#181;l of NaCl, and load the cells into a 1 ml insulin syringe with a 30G needle. Lightly vortex the solution before loading the syringe to ensure all monocytes are injected. Always handle cells on ice to avoid heat-mediated attachment and activation of monocytes.</li>
</ol>
2.2 Restraining
<ol>
	<li>Avoid disturbing other mice in the cage when choosing a mouse for intravenous injection.</li>
	<li>Place the mouse into the restrainer. Avoid too much pressure and be careful with the hind limbs. (Figure 4) 
	Note: Alternatively, use general anesthesia to guarantee stress free handling of the animals.</li>
	<li>Ensure that the mouse has space for breathing before closing the restrainer.</li>
</ol>
2.3 Injection
<ol>
	<li>Disinfect the injection site with disinfection agent. Spray on the tail of the mouse and let stand for at least 1 min.</li>
	<li>Stop venous blood flow of the tail by applying gentle pressure to the lateral tail side above the injection site, for better visibility of the veins.</li>
	<li>Turn the tail 90 degrees, before injection. (Figure 5)</li>
	<li>Inject at a 45 degree angle. Inject slowly and no more than 5 &#181;l per g to avoid harming animals.</li>
	<li>If there is a blister, which is a sign of a failed injection, discontinue immediately and repeat the injection more proximally.</li>
	<li>Stop the bleeding at the injection side by applying gentle pressure for about 1 min.</li>
	<li>At the end of the procedure, open the retractor and place the mouse in its cage.</li>
	<li>Observe the mouse for 20 min to ensure that the animal was not harmed by the injection and sternal recumbency is maintained. Extend the time of observation until the mouse regains sufficient consciousness. Return the mouse to the company of other animals only after it has fully recovered.</li>
</ol>

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The authors declare that they have no competing financial interests.

We describe a simple and cost-effective method to isolate large amounts of murine monocytes from bone marrow. In comparison to other protocols using peripheral blood, which obtain monocyte yields5 of 1.4 x106, we are able to obtain higher yields of 11 x 106 &#177; 3 x 106 monocytes from a single donor mouse.
When considering challenges with this method, it is important to mention the potential for contamination when working under non-sterile conditions. If rinsing and the following washing steps are not followed properly, contamination by bacteria and fungi is likely. Additionally, bone marrow consists of heterogeneous cell suspensions, including granulocytes, lymphocytes and erythroid cells. Normally these cells vanish between day 2 and 3 of cultivation due to the lack of growth factors. After growth factor driven differentiation, macrophages are the major source of contamination. To keep the number of macrophages low and to minimize the differentiation of monocytes to macrophages, the use of ultra-low attachment plates and the washing steps mentioned above are critical.
It is difficult to identify a marker that is exclusively monocyte-specific. Interference between markers necessitates the use of more than one marker, and there is variability in the granularity and size of cells in analyzing cell suspensions. The markers and descriptions shown in Figures 1-3 are good alternatives for representative cell types in culture. It can be difficult to distinguish the various cell types, especially after day 5. Therefore it is critical to use other markers like CD115 and F4/80.
Considering the expansion of bone marrow cells under adhesion-free conditions, macrophage differentiation can be prolonged and undifferentiated monocytes can accumulate. Macrophage differentiation indicates contamination; however, due to their adhesive nature even on ultra-low-attachment cultivation surfaces, they can be easily discarded while only harvesting non-adhesive cells from culture. In previous experiments1,3, contamination by macrophages has not led to serious clinical side effects. Histological workup of organs and muscles of mice after cell transplantation showed that injected macrophages assimilated in organs like spleen, liver or lung. The assimilation of macrophages in these organs demonstrated no observable clinical effects in mice. The molecular mechanism triggered by these macrophages can be interesting questions for further investigations.
Monocytes as well as macrophages affect arteriogenesis in peripheral arterial disease, especially when applied systemically. Clinical side effects such as embolism or massive systemic inflammation could not be observed even after injection of more than 2.5 million monocytes, but these cells can trigger inflammation and potentially cause severe systemic effects. In human trials, these effects are likely to have clinical significance. The triggering of arteriogenesis can affect tumor growth or diabetic retinopathy, and such possible systemic effects of injected cell solutions should be explored in future studies.
Previous publications9 describe differences between the characteristics of peripheral blood- and bone marrow-derived monocytes, which explain why the properties of monocytes from bone marrow can differ from those isolated from peripheral blood cells.
The method of isolation is of clinical interest as well. Drawing blood instead of extracting human bone marrow is more practical in the context of clinical medicine. It is important to look after the welfare of the animals and to apply analgesia if needed. For intravenous injections, it is critical to practice handling both the animal and the syringe at the same time. If a single animal is undergoing multiple injections, the quality of the veins and the skin of the tail suffer.
Despite these disadvantages, this method is very useful to study the behavior and characteristics of monocytes. Experiments in the field of atherosclerosis, immunology or inflammation can benefit from this method. Only 30 min&#160;are required from the extraction of bone marrow to the seeding of cells on 6-well ultra-low-attachment surface plates. Ethical concerns are minimized, because the high yield of this protocol minimizes the number of animals required as cell donors. This new method will be helpful for many studies involving monocytes.
We have been focused on therapeutic augmentation of collateral vessel growth via transplantation of monocytes. The multifactorial nature of this process may explain why single factor approaches for augmentation of arteriogenesis have generated mixed results13,14 . This has led to the investigation of cell-based therapies. Autologous bone marrow-derived stem and progenitor cells have been identified as potential cells for transplantation, but only moderate clinical benefits have been reported15. Besides research on dosage, isolation methods, and administration routes, further research is still needed to determine the optimal cell type. A disadvantage of autologous cell transplantation could be their inability to activate the innate and/or adaptive immune response, and both are critical for arteriogenesis. Increasing local inflammation may represent the best stimulus for arteriogenesis16.
Intravenous injection of monocytes is a common method for cell transplantation within mouse models if systemic drug delivery is desired. Because of systemic effects, side effects can occur as well. Ensure that the vein is targeted for injection. It is common to confuse arteries and veins, especially when injecting heavily pigmented mice. Arterial injection can cause emboli, which lead to systemic issues in circulation.

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.

The cell solution extracted from the murine bone marrow consists of various cell types. The major cell types are lymphocytes, granulocytes and monocytes. Cell types can be estimated by size and granularity, which is shown in Figure 1 for both native suspensions and cells harvested after 5 days of differentiation. Note the shifting cellular composition during cultivation. However, accurate classification of populations must rely on distinctive expression of cellular markers.
The most important marker used to identify cells of the MPS-System is CD115, which functions as the M-CSF receptor and is specifically expressed on monocyte progenitors, monocytes and macrophages. Therefore, it might not be used to identify distinctive MPS-subsets, but can reliably estimate the absolute amount of cells driven into the monocyte/macrophage differentiation. See Figure 2 for a timeline of CD115 expression during differentiation.
The maturity marker F4/80 is useful to differentiate juvenile monocytes, mature monocytes, and macrophages. Macrophages show strong expression of F4/80 in comparison to juvenile monocyte progenitors, which are negative for the marker. Mature monocytes show intermediate expression of F4/80. A combination of CD115 and F4/80 markers therefore represents a feasible method for quantifying and monitoring cell differentiation (see Figure 3).
The combination of CD115 and F4/80 is sufficient for routine culture observation and quality control prior to application of cells. A more detailed examination of the bone-marrow-derived monocytes at day 5 of cultivation confirms their structural and functional properties in concordance to those of peripheral blood monocytes 12.
It is important to fix the mouse carefully in the restrainer, avoiding tension on the tail. Restrict freedom of movement as much as possible without inhibiting breathing to facilitate the injection.
<img alt="Figure 1" src="/files/ftp_upload/52347/52347fig1highres.jpg" /> 
Figure 1. Left panel: Composition of cell types at day 1 in culture. Cell types include lymphocytes, monocytes and granulocytes. Right panel: Note the shifting cellular composition during cultivation. Cell population includes monocytes and macrophages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52347/52347fig2highres.jpg" /> 
Figure 2.&#160;Timeline of CD115 expression during differentiation. Note that &#62;95% of all cells are CD115 positive after 5 days of differentiation, indicating that the vast majority of cells are either monocytes or macrophages. Classification can be based on cell size and granularity, but specific cellular markers are more reliable. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52347/52347fig3highres.jpg" /> 
Figure 3. Increasing expression of monocyte/macrophage maturity marker F4/80. Day 5: Note the appearance of a population with intermediate F4/80 expression, which is consistent with monocyte phenotype. Day 7: Note the right-shift of F4/80 expression, consistent with macrophage maturation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52347/52347fig4highres.jpg" /> 
Figure 4. Mouse fixed in restrainer for tail vein injection. After carefully restraining the mouse, rotate the tail 90 degrees until the veins are visible on the dorsal side. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52347/52347fig5highres.jpg" /> 
Figure 5. Schematic cross section of the mouse tail. The picture illustrates the location of vessels within the mouse tail. The arteries (red) are located on the ventral and dorsal side, and the veins are on the lateral side of the tail. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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).

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.6K 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

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.

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 ...

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.

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.

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.

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

Medicine

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.

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.

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

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

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.

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

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.

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.

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 ...

Intrafemoral Injection for Bone Marrow Study in Live Mice

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Secure a euthanized mouse supine on a foam board with its limbs outstretched, then, clean the legs with 70% ethanol. Next, make a shallow cut about 2-millimeter deep to remove the skin and fur from the surface of each hind leg. Then, dissect the tissues to access the tibias and femurs.
To remove the tibias and femurs, cut the bone and muscles just below the hip joint and above the ankles. Then, remove as much of the muscle tissue from the bones as possible. Next, clean the bones with 70% ethanol and allow it to evaporate for 1 minute, then, place the bones in a 6-well plate filled with bone flush medium.
In the plate, trim 1 millimeter from either end of the femurs so that their inner cavities are exposed. Make certain a 26-gauge needle can access the cavities. Next, separate the tibia and femur from the knee joint. Then, insert the needle attached to a 10-milliliter syringe into a bone cavity and collect the bone marrow.
Pool the bone marrow from all 4 bones into a 50-milliliter conical tube with 5 milliliters of bone flush medium. Pipette the medium and marrow up and down several times, or vortex at a slow speed to gently disperse clumps in the suspension. Strings of marrow should be broken up into small flecks of marrow less than half a millimeter long.
Next, top off the tube to 50 milliliters with bone flush medium and transfer it into a 75-square-centimeter tissue culture flask. Incubate the bone marrow for an hour. During the incubation, the matured cells will adhere to the flask and the desired hematopoietic progenitors will remain in suspension. Transfer the suspension to a 50-milliliter conical tube, and spin down the cells into a pellet.
Next, discard the supernatant, and resuspend the cell pellet in 5 milliliters of MCSF culture medium. Then, adjust the cell density to 500,000 per milliliter and load 30 milliliters into a new 75-square-centimeter flask. Continue culturing the cells and on days 4, 7, and 10, verify that the cells are adherent, and slightly branched using an inverted phase contrast brightfield microscope.
When checking the cells, discard the medium containing the non-adherent cells. Then, wash the adherent cells with IMDM one time, and add 30 milliliters of fresh MSCF culture medium back into the flask. On day 10, replace the medium with 8 milliliters of enzyme-free EDTA-based cell dissociation buffer to collect the cells. Incubate the cells briefly, then, scrape the cells gently and verify that they&#39;ve released using a microscope.
Once the cells are released, transfer the buffer with the cells to a 50-milliliter conical tube, then rinse the flask three times with 5 milliliters of IMDM to collect more cells. Pool all the collections, centrifuge them, and resuspend the pellet in 3 milliliters of MCSF. Now, count the cells and adjust their concentration to 1 million per milliliter.
Then, plate 100 microliters of cell suspension per well in a 96-well flat-bottom plate. The entire plate should be fillable. Culture the cells for about an hour until they are adherent. Then, stimulate them with different concentrations of LPS or intravenous immunoglobulin or both, prepared in IMDM. Perform all the manipulations in duplicate or triplicate and be certain to keep untreated controls, then, incubate the cells for 24 hours and analyze the supernatants.

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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