Name: isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method
Uploaded: 2022-07-13T15:00:00
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...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm2 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 &#956;g/test,1.675 mg/Ml</td>
		</tr>
		<tr>
			<td>Anti-CD11b/c</td>
			<td>Absin</td>
			<td>abs124232</td>
			<td>&#160;1&#956;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-&#946;-actin</td>
			<td>Beyotime</td>
			<td>&#160;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&#39;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 ...

Research

JoVE Journal

Medicine

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...