Name: isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method
Uploaded: 2022-07-13T15:00:00.000+00:00
Duration: 4 min 19 s
Description: Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

This work was supported by the Natural Science Foundation of Zhejiang Province (grant no. LY19H060001) and the Zhejiang Traditional Chinese Medicine Science and Technology Plan Project (no. 2022ZB093).

All experiments in this study were conducted in accordance with the animal experiment guidelines of the Zhejiang Chinese Medical University Laboratory Animal Research Center (Approval No: IACUC-20181029-11).
1. Cell extraction
<ol>
	<li>Put the Sprague-Dawley rats (SD rats, 1-10 days old, male or female) in the euthanasia cages filled with CO2 at a balanced rate of 30%-70% container volume/minute. After the rats lose consciousness (20-60 min), euthanize the rat by cervical dislocation to ensure a painless death.</li>
	<li>Immerse the rats in 75% alcohol for 10 min for disinfection.</li>
	<li>Carefully remove all the limbs of the rat with scissors and forceps, aspirate with PBS using a pipette and flush the blood adhering to the limbs.</li>
	<li>Add 5 mL of penicillin/streptomycin solution into 500 mL of DMEM and mix well. Take a 50 mL sterile centrifuge tube, add 5 mL of FBS and 45 mL of DMEM medium, and mix thoroughly to obtain a 10% FBS DMEM containing 1% penicillin/streptomycin solution. Add 2 mL of the above culture medium into a 5 mL tube.</li>
	<li>Transfer the limb bones into the 5 mL tube. Use scissors to cut the limb bones in the tube into small pieces (1-3 mm) and mix the homogenate to re-suspend the bone marrow cells into the culture medium. Stand for 5 min until the tissue fragments settled to the bottom of the tube.</li>
	<li>Add 10&#160;mL of 10% FBS DMEM into a 100 mm culture dish, and then transfer the supernatant into the culture dish. When aspirating the supernatant, avoid transferring the tissue debris into the culture dish.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for approximately 24 h. After this incubation time, the majority of mesenchymal stem cells will adhere to the culture dish wall and grow slowly, while most BMMs will still be suspended in the culture medium.</li>
	<li>Transfer the cell suspension in the 100 mm culture dish to a new 25 cm2 flask and continue to cultivate the cells at 37 &#176;C and 5% CO2 for an additional 24 h. BMMs will adhere to the flask wall at 24-48 h of culture. Remove the old medium carefully and replace with fresh medium after BMMs have adhered to the flask wall.</li>
	<li>Sub-culture when the cells in the flask reached 80%-90% confluency. 
	&#8203;NOTE: All the cells were cultured at 37 &#176;C and 5% CO2. During culture, the cell morphology was gradually unified. Cells were large, irregularly shaped, radially growing and attached to the flask in a form of disc, where multiple nuclei could be observed.</li>
</ol>
2. FACS staining of the cell
<ol>
	<li>Trypsin digest using 1-2 mL of commercial trypsin at 37 &#176;C and 5% CO2 for 5 min and count secondary adherent cells to ensure a final cell count of 100,000/sample (count cells with Neubauer hemocytometer).</li>
	<li>Divide the cells into three groups (500 &#181;L of PBS contains 100,000 cells): (1) the blank control group containing unstained cells; (2) the isotype control group; and (3) the experimental group (CD11b/c staining).</li>
	<li>Incubate primary antibodies (no antibody for the blank control group; anti-CD11 isotype control for the isotype control group, 0.6 &#181;L of antibody/sample, 1 &#181;g/sample; anti-CD11b/c for the experimental group, 1 &#181;L of antibody/sample, 1 &#181;g/sample) on ice for 30 min.</li>
	<li>Centrifuge at 300-350 x g for 5 min, discard the supernatant, and resuspend the cells with 500 &#181;L of PBS. Repeat the above procedure once to ensure that the unbound primary antibodies are washed away.</li>
	<li>Incubate the corresponding secondary antibody (no antibody for the blank control group; goat anti-rabbit IgG for the isotype control and the experimental groups, 0.25 &#181;L antibody/sample, 1:2,000) on ice in the dark for 30 min.</li>
	<li>Centrifuge at 300-350 x g for 5 min, discard the supernatant, and resuspend the cells with 500 &#181;L of PBS. Repeat the above procedure once to ensure that the unbound second antibodies are washed away.</li>
	<li>Detect the CD11b/c positive cells by flow cytometry (10,000 cells/sample) and analyze the data by software (e.g., FlowJo 7.5). 
	&#8203;NOTE: To obtain the final percentage of CD11b/c+ cells, the following formula was used: Percentage of CD11b / c + cells in the experimental group - the percentage of CD11 + cells in the isotype control group.</li>
</ol>
3. Wright-Giemsa staining
<ol>
	<li>Seed the adherent secondary cells that have been passaged three times onto 35 mm2 cell climbing sheets (1 &#215; 106 cells/well) and culture at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Discard the culture medium and wash thrice with PBS.</li>
	<li>Add Wright-Giemsa dye solution (0.5 mL-0.8 mL) to the cell climbing sheet for 1 min.</li>
	<li>Mix the dye with distilled water (0.5 mL-0.8 mL) with a cotton swab, and stand for 10 min.</li>
	<li>Wash the dye solution with distilled water, and then dry for 1-3 min. Observe under a microscope.</li>
</ol>
4. TRAP staining
<ol>
	<li>Seed the adherent secondary cells that have been passaged three times onto 35 mm2 cell climbing sheets (1 &#215; 106 cells/well) and culture at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Replace the old medium with the 10% FBS DMEM or osteoclast induction medium supplemented with 50 ng/mL receptor activator of nuclear factor-&#954;B ligand (RANKL) and 30 ng/mL of macrophage colony-stimulating factor (M-CSF), and culture at 37 &#176;C and 5% CO2 for an additional 7 days.</li>
	<li>Stain the cells with the TRAP staining kit according to manufacturer&#39;s protocol and observe under a microscope. 
	&#8203;NOTE: TRAP-positive cells were defined as osteoclasts, which were purple under a light microscope. The number of TRAP-positive cells was measured using ImageJ software.</li>
</ol>
5. Western blot
<ol>
	<li>Seed the adherent secondary cells that have been passaged three times on 35 mm2 cell climbing sheets (1 &#215; 106 cells/well) and culture for 24 h.</li>
	<li>Replace the old medium with fresh 10% FBS DMEM or osteoclast induction medium (50 ng/mL of RANKL and 30 ng/mL of M-CSF) and culture for an additional 7 days at 37 &#176;C and 5% CO2.</li>
	<li>Extract total cellular proteins with RIPA buffer, separate by 10% SDS-PAGE, and transfer to polyvinylidene fluoride membranes18,19.</li>
	<li>Block with 5% skimmilk powder (25 mL) for 2 h and wash three times, for 10 min each time, with TBS-Tween 20 (TBST).</li>
	<li>Incubate primary antibodies at 4 &#176;C overnight (anti-&#946;-actin, anti-TRAP, and anti-cathepsin K; all primary antibodies were diluted 1:1,000, 10 mL diluted antibody/band), and wash thrice with TBST.</li>
	<li>Incubate the secondary antibody (goat anti-rabbit IgG, 1:2,000 dilution, 10 mL diluted antibody/band) at room temperature for 2 h, and wash thrice with TBST. Visualize the protein bands using a developing solution. 
	NOTE: The expression levels of the above proteins were normalized to &#946;-actin.</li>
</ol>

<ol>
	<li>Jakubzick, C. V., Randolph, G. J., Henson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+differentiation+and+antigen-presenting+functions.">Monocyte differentiation and antigen-presenting functions.</a> Nature Reviews. Immunology. 17 (6), 349-362 (2017).</li><li>Locati, M., Curtale, G., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity,+mechanisms,+and+significance+of+macrophage+plasticity.">Diversity, mechanisms, and significance of macrophage plasticity.</a> Annual Review of Pathology. 15 (1), 123-147 (2020).</li><li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+and+activation.">Osteoclast differentiation and activation.</a> Nature. 423 (6937), 337-342 (2003).</li><li>Ono, T., Nakashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+osteoclast+biology.">Recent advances in osteoclast biology.</a> Histochemistry and Cell Biology. 149 (4), 325-341 (2018).</li><li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/&#223;-catenin-mediated+p53+suppression+is+indispensable+for+osteogenesis+of+mesenchymal+progenitor+cells.">Wnt/&#223;-catenin-mediated p53 suppression is indispensable for osteogenesis of mesenchymal progenitor cells.</a> Cell Death &amp; Disease. 12 (6), 521-534 (2021).</li><li>Yu, Q., Zhao, B., He, Q., Zhang, Y., Peng, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-206+is+required+for+osteoarthritis+development+through+its+effect+on+apoptosis+and+autophagy+of+articular+chondrocytes+via+modulating+the+phosphoinositide+3-kinase/protein+kinase+B-mTOR+pathway+by+targeting+insulin-like+growth+factor-1.">microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1.</a> Journal of Cellular Biochemistry. 120 (4), 5287-5303 (2019).</li><li>Li, Z., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+for+investigating+how+bone+marrow+adipocytes+influence+bone+and+hematopoietic+cellularity.">Preclinical models for investigating how bone marrow adipocytes influence bone and hematopoietic cellularity.</a> Best Practice &amp; Research. Clinical Endocrinology &amp; Metabolism. 35 (4), 101547-101560 (2021).</li><li>Horowitz, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+adipocytes.">marrow adipocytes.</a> Adipocyte. 6 (3), 193-204 (2017).</li><li>Maridas, D. E., Rendina-Ruedy, E., Le, P. T., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+differentiation+of+bone+marrow+stromal+cells+and+osteoclast+progenitors+frommice.">Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors frommice.</a> Journal of Visualized Experiments. (131), e56750 (2018).</li><li>Schyns, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-classical+tissue+monocytes+and+two+functionally+distinct+populations+of+interstitial+macrophages+populate+the+mouse+lung.">Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung.</a> Nature Communications. 10 (1), 3964-3980 (2019).</li><li>Atif, S. M., Gibbings, S. L., Jakubzick, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+interstitial+macrophages+from+the+lungs+using+different+digestion+enzymes+and+staining+strategies.">Isolation and identification of interstitial macrophages from the lungs using different digestion enzymes and staining strategies.</a> Methods in Molecular Biology. 1784, 69-76 (2018).</li><li>Scheven, B. A., Milne, J. S., Robins, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sequential+culture+approach+to+study+osteoclast+differentiation+from+nonadherent+porcine+bone+marrow+cells.">A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 34 (7), 568-577 (1998).</li><li>Bradley, E. W., Oursler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+culture+and+resorption+assays.">Osteoclast culture and resorption assays.</a> Methods in Molecular Biology. , 19-35 (2008).</li><li>Yu, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+myeloid+cells+in+human+blood,+bronchoalveolar+lavage,+and+lung+tissues.">Flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues.</a> American Journal of Respiratory Cell and Molecular Biology. 54 (1), 13-24 (2016).</li><li>Gibbings, S. L., Jakubzick, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+consistent+method+to+identify+and+isolate+mononuclear+phagocytes+from+human+lung+and+lymph+nodes.">A consistent method to identify and isolate mononuclear phagocytes from human lung and lymph nodes.</a> Methods in Molecular Biology. 1799, 381-395 (2018).</li><li>Jacquin, C., Gran, D. E., Lee, S. K., Lorenzo, J. A., Aguila, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+multiple+osteoclast+precursor+populations+in+murine+bone+marrow.">Identification of multiple osteoclast precursor populations in murine bone marrow.</a> Journal of Bone and Mineral Research. 21 (1), 67-77 (2006).</li><li>Gibbings, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+unique+interstitial+macrophages+in+the+murine+lung+at+steady+state.">Three unique interstitial macrophages in the murine lung at steady state.</a> American Journal of Respiratory Cell and Molecular Biology. 57 (1), 66-76 (2017).</li><li>Higashi, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-speed+western+blot+using+immunoreaction+enhancing+technology.">Ultra-high-speed western blot using immunoreaction enhancing technology.</a> Journal of Visualized Experiments. (163), e61657 (2020).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> Journal of Visualized Experiments. 20 (16), 759 (2008).</li><li>Yin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycyrrhizic+acid+suppresses+osteoclast+differentiation+and+postmenopausal+osteoporosis+by+modulating+the+NF-&#954;B,+ERK,+and+JNK+signaling+pathways.">Glycyrrhizic acid suppresses osteoclast differentiation and postmenopausal osteoporosis by modulating the NF-&#954;B, ERK, and JNK signaling pathways.</a> European Journal of Pharmocology. 859, 172550 (2019).</li><li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRc17+controls+BMSC+senescence+via+mitophagy+and+inhibits+the+therapeutic+effect+of+BMSCs+on+ovariectomy-induced+bone+loss.">LRRc17 controls BMSC senescence via mitophagy and inhibits the therapeutic effect of BMSCs on ovariectomy-induced bone loss.</a> Redox Biology. , (2021).</li><li>Jin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioacetamide+promotes+osteoclast+transformation+of+bone+marrow+macrophages+by+influencing+PI3K/AKT+pathways.">Thioacetamide promotes osteoclast transformation of bone marrow macrophages by influencing PI3K/AKT pathways.</a> Journal of Orthopedic Surgery and Research. 17 (1), 53-63 (2022).</li></ol>

The authors declare that they have no competing interests.

Osteoclasts are one of the most significant cell types involved in the occurrence and development of bone diseases, as well as one of the primary objects of bone disease research20. Monocyte/macrophages can differentiate into osteoclasts. Since mononuclear macrophages (RAW264.7 cells) are too expensive to buy and are easily activated during culture, it is difficult to perform in vitro differentiation experiments using this cell line. Although several methods have been developed for extrac...

It has been reported that monocyte-macrophage lineage cells existing in the bone marrow can differentiate into blood monocytes and tissue macrophages1,2. The above cells, which can differentiate into osteoclasts to balance bone growth and development, are commonly used as a cell model to induce osteoclasts in vivo3,4. Bone marrow is a special tissue containing several different types of cells, which include but are not only limited to bone marrow mesenchymal stem cells, bone marrow monocyte-macrophage cells (BMMs), hematopoietic stem cells, endothelial cells, and immune cells. In fact, several previous studies suggested that adherent cells rushed out of the bone marrow cavity of the long bone could differentiate into osteoblasts, osteoclasts, chondrocytes, or adipocytes5,6,7,8. Although, different isolation and culture methods have been used to produce different homogeneous cell populations, there are still great challenges in isolating and culturing BMMs from a variety of different cell types.
Several methods have been developed to extract bone marrow mononuclear macrophages (BMSCs). However, the majority of these methods are complex9,10,11. For example, density gradient centrifugation requires a specialized kit and the operation is time-consuming and cumbersome. This method is suitable for the isolation of BMMs from high-volume blood samples, but not from bone marrow samples9,12,13. In addition, extracting tissue samples using collagenase digestion is a complex and time-consuming procedure; this method is not recommended for the isolation of BMMs from bone marrow samples14,15. In addition, although flow separation can result in highly purified monocyte/macrophage populations, it requires very large sample sizes and high instrument and equipment requirements10,16. Additionally, the microbead enrichment method is extremely expensive and is not feasible in a general laboratory17.
Therefore, in the current study a convenient, fast, and cheap method was proposed for the isolation of mononuclear macrophages from the bone marrow. Bone marrow cells adhered for different time points were used to isolate BMMs using a secondary adherence method. BMMs extracted with the above method could induce the formation of osteoclasts in vitro, thus providing a simple and convenient cell model for the future study of osteoporosis in vitro.

<table><tbody><tr><td>35 mm&lt;sup&gt;2&lt;/sup&gt; cell climbing slices</td><td>NEST Biotechnology</td><td>80102</td><td></td></tr><tr><td>Anti-cathepsin K</td><td>Abcam</td><td>ab19027</td><td>1:1,000</td></tr><tr><td>Anti-CD11 isotype control</td><td>Abcam</td><td>ab172730</td><td>1 &amp;mu;g/test,1.675 mg/Ml</td></tr><tr><td>Anti-CD11b/c</td><td>Absin</td><td>abs124232</td><td>&amp;nbsp;1&amp;mu;g/test, 1 mg/mL</td></tr><tr><td>Anti-TRAP</td><td>Abcam</td><td>ab191406</td><td>1:1,000</td></tr><tr><td>Anti-&amp;beta;-actin</td><td>Beyotime</td><td>&amp;nbsp;AF5003</td><td>1:1,000</td></tr><tr><td>Cell climbing slices</td><td>NEST Biotechnology</td><td>80102</td><td></td></tr><tr><td>Cell culture dish</td><td>corning</td><td>430167</td><td></td></tr><tr><td>Cell culture flask</td><td>corning</td><td>430168</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Gibco</td><td>C11995500BT</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>10099141C</td><td></td></tr><tr><td>Goat anti-rabbit IgG</td><td>Abcam</td><td>ab150077</td><td>for IF, 1:2,000</td></tr><tr><td>goat anti-rabbit IgG</td><td>Abcam</td><td>ab6721</td><td>for WB, 1:2,000</td></tr><tr><td>M-CSF</td><td>Pepro tech</td><td>400-28</td><td></td></tr><tr><td>PBS</td><td>Biosharp</td><td>BL302A</td><td></td></tr><tr><td>RANKL</td><td>Pepro tech</td><td>400-30</td><td></td></tr><tr><td>SD rat</td><td>Shanghai SLAC Laboratory Animal Co, Ltd</td><td></td><td>1-10 days old</td></tr><tr><td>SDS-PAGE gel preparation kit</td><td>Solarbio</td><td>P1200</td><td></td></tr><tr><td>TRAP/ALP Staining Kit</td><td>Wako</td><td>294-67001</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Biosharp</td><td>BL512A</td><td></td></tr><tr><td>Wright-Giemsa solution</td><td>Keygen Biotech</td><td>KGA225-1</td><td></td></tr></tbody></table>

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.

The secondary adherent cell population was stable and uniform. With the continuous cell proliferation, the majority of cells became larger, with irregular shape and grew into a radial adherent disk (Figure 2C,D). Flow cytometry showed that the percentage of cells expressing CD11b/c, a molecular marker on the surface of monocyte-macrophage lineage cells, was approximately 37.94% (Figure 2A,B). To ...

Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

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

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 ...

isolation-monocyte-macrophage-lineage-cells-from-rat-bones-secondary

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JoVE Journal

Medicine

3.5K Views.  Zhejiang Chinese Medical University. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. 

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Immunology and Infection

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.

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 ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Bioengineering

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

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 and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Biology

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...