Name: Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method (Video) | JoVE Journal
Uploaded: 2022-07-13T15:00:00.000+00:00
Duration: 4 min 19 s
Description: Learn the secondary adherence method to isolate bone marrow monocyte-macrophage cells from SD rats for osteoclast differentiation studies. This protocol provides a reliable model for osteoporosis research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((Monocyte+differentiation+and+antigen-presenting+functions%5BTitle%5D)+AND+%22Nature+Reviews.+Immunology%22%5BJournal%5D)">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/pubmed?term=((Diversity%2C+mechanisms%2C+and+significance+of+macrophage+plasticity%5BTitle%5D)+AND+%22Annual+Review+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Osteoclast+differentiation+and+activation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Recent+advances+in+osteoclast+biology%5BTitle%5D)+AND+%22Histochemistry+and+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Wnt%2F%C3%9F-catenin-mediated+p53+suppression+is+indispensable+for+osteogenesis+of+mesenchymal+progenitor+cells%5BTitle%5D)+AND+%22Cell+Death+%26+Disease%22%5BJournal%5D)">Wnt/ß-catenin-mediated p53 suppression is indispensable for osteogenesis of mesenchymal progenitor cells.</a> Cell Death & 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/pubmed?term=((microRNA-206+is+required+for+osteoarthritis+development+through+its+effect+on+apoptosis+and+autophagy+of+articular+chondrocytes+via+modulating+the+phosphoinositide+3-kinase%2Fprotein+kinase+B-mTOR+pathway+by+targeting+insulin-like+growth+factor-1%5BTitle%5D)+AND+%22Journal+of+Cellular+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Preclinical+models+for+investigating+how+bone+marrow+adipocytes+influence+bone+and+hematopoietic+cellularity%5BTitle%5D)+AND+%22Best+Practice+%26+Research.+Clinical+Endocrinology+%26+Metabolism%22%5BJournal%5D)">Preclinical models for investigating how bone marrow adipocytes influence bone and hematopoietic cellularity.</a> Best Practice & Research. Clinical Endocrinology & Metabolism. 35 (4), 101547-101560 (2021).</li>
<li>Horowitz, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((marrow+adipocytes%5BTitle%5D)+AND+%22Adipocyte%22%5BJournal%5D)">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/pubmed?term=((Isolation%2C+culture%2C+and+differentiation+of+bone+marrow+stromal+cells+and+osteoclast+progenitors+frommice%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Non-classical+tissue+monocytes+and+two+functionally+distinct+populations+of+interstitial+macrophages+populate+the+mouse+lung%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+identification+of+interstitial+macrophages+from+the+lungs+using+different+digestion+enzymes+and+staining+strategies%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((A+sequential+culture+approach+to+study+osteoclast+differentiation+from+nonadherent+porcine+bone+marrow+cells%5BTitle%5D)+AND+%22In+Vitro+Cellular+%26+Developmental+Biology.+Animal%22%5BJournal%5D)">A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells.</a> In Vitro Cellular & 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/pubmed?term=((Osteoclast+culture+and+resorption+assays%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Flow+cytometric+analysis+of+myeloid+cells+in+human+blood%2C+bronchoalveolar+lavage%2C+and+lung+tissues%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+Cell+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((A+consistent+method+to+identify+and+isolate+mononuclear+phagocytes+from+human+lung+and+lymph+nodes%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Identification+of+multiple+osteoclast+precursor+populations+in+murine+bone+marrow%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">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/pubmed?term=((Three+unique+interstitial+macrophages+in+the+murine+lung+at+steady+state%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+Cell+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Ultra-high-speed+western+blot+using+immunoreaction+enhancing+technology%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Immunoblot+analysis%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Glycyrrhizic+acid+suppresses+osteoclast+differentiation+and+postmenopausal+osteoporosis+by+modulating+the+NF-%CE%BAB%2C+ERK%2C+and+JNK+signaling+pathways%5BTitle%5D)+AND+%22European+Journal+of+Pharmocology%22%5BJournal%5D)">Glycyrrhizic acid suppresses osteoclast differentiation and postmenopausal osteoporosis by modulating the NF-κB, 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/pubmed?term=((LRRc17+controls+BMSC+senescence+via+mitophagy+and+inhibits+the+therapeutic+effect+of+BMSCs+on+ovariectomy-induced+bone+loss%5BTitle%5D)+AND+%22Redox+Biology%22%5BJournal%5D)">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/pubmed?term=((Thioacetamide+promotes+osteoclast+transformation+of+bone+marrow+macrophages+by+influencing+PI3K%2FAKT+pathways%5BTitle%5D)+AND+%22Journal+of+Orthopedic+Surgery+and+Research%22%5BJournal%5D)">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

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 ...

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

Research

JoVE Journal

Medicine

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.

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

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

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).

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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.

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

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.

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

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.

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

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.

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

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

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

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.

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

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.

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

Learn the secondary adherence method to isolate bone marrow monocyte-macrophage cells from SD rats for osteoclast differentiation studies. This protocol provides a reliable model for osteoporosis research.

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

Explore More Videos