Name: economical and efficient protocol for isolating and culturing bone marrow-derived dendritic cells from mice
Uploaded: 2022-07-01T12:30:00
Description: Watch this Scientific Journal Video about Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice at JoVE.com

This work was supported by Program of Tianjin Science and Technology Plan (20JCQNJC00550), Tianjin Health Science and Technology Project (TJWJ202021QN033 and TJWJ202021QN034).

All procedures were approved by the Nanjing Medical University Animal Care and Use Committee.
1. Isolation of bone marrow and preparation of BM cells
<ol>
	<li>Sacrifice C57BL/6 mice (18-22 g, 6-8 weeks old) via CO2 asphyxiation. Fix the mouse on the mouse operating table. Disinfect the surfaces with 70% ethanol.</li>
	<li>Cut the skin of the leg to expose the muscles and femoral artery. Clamp and tear off the femoral artery using two forceps, then pull the proxim...

<ol>
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Humans and mice have different DC subsets, including classical DCs (cDCs, including cDC1s and cDC2s) plasmacytoid DCs (pDCs), and monocyte-derived DCs (MoDCs)9,10,11. It is generally accepted that cDC1s regulate cytotoxic T lymphocyte (CTL) responses to intracellular pathogens and cancer, and cDC2s regulate immune responses to extracellular pathogens, parasites, and allergens12. A significant number of DC...

Dendritic cells (DCs) are the most powerful antigen-presenting cells (APCs) for activating na&#239;ve T cells and inducing specific cytotoxic T lymphocyte (CTL) responses against infectious diseases, allergy diseases, and tumor cells1,2,3. DCs are the primary link between innate immunity and adaptive immunity and play an essential role in immunological defense and the maintenance of immune tolerance. In the last 40 years, many researchers have sought to define the subsets of DCs and their functions in inflammation and immunity. As per those studies, DCs develop along the myeloid and lymphoid lineages from bone marrow cells. Tumor vaccines have gained significant milestones in recent years and have a promising future. Mechanically, tumor vaccines modulate the immune response and prevent tumor growth by activating cytotoxic T lymphocytes using tumor antigens. The vaccine based on DCs plays an important role in tumor immunotherapy and has been identified as one of the most promising anti-tumor therapies1,4. In addition, DCs have been widely used in the testing of new molecular-targeted drugs and immune checkpoint inhibitors5.
Researchers urgently need a high number of high-purity DCs to further study the role of DCs. However, DCs are rare in various tissues and blood, accounting for only 1% of blood cells in humans and animals. In vitro culture of bone marrow dendritic cells (BMDC) is an important method for obtaining large amounts of DC cells. Meanwhile, The Lutz protocol for generating DCs from bone marrow has been widely used by researchers6. Although the protocol is effective in obtaining DC cells, it is complex and expensive, involving the addition of high concentrations of cytokines and the lysis of red blood cells.
In this study, we report a method for isolating almost all bone marrow cells from mouse bone marrow (BM) and inducing differentiation into BMDC after 7-9 days of incubation in vitro, with a lower concentration of GM-CSF and IL-4. This procedure only takes 10 min to harvest almost all bone marrow cells and to suspend them in a complete medium. In brief, we provide an efficient and cost-effective culturing method for BMDC in this research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Solarbio</td>
			<td>M8211</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-MHC II</td>
			<td>Biolegend</td>
			<td>116417</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10100</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD80</td>
			<td>Biolegend</td>
			<td>104707</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Solarbio</td>
			<td>P1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Percp/cy5.5-CD11c</td>
			<td>Biolegend</td>
			<td>117327</td>
			<td></td>
		</tr>
		<tr>
			<td>PRMI-1640</td>
			<td>Thermo</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse GM-CSF</td>
			<td>Solarbio</td>
			<td>P00184</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-4</td>
			<td>Solarbio</td>
			<td>P00196</td>
			<td></td>
		</tr>
		<tr>
			<td>TruStain Fc PLUS (anti-mouse CD16/32) Antibody</td>
			<td>Biolegend</td>
			<td>156603</td>
			<td></td>
		</tr>
	</tbody>
</table>

economical and efficient protocol for isolating and culturing bone marrow-derived dendritic cells from mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

The 1 x 107-1.7 x 107 cells were extracted from two femurs and were re-suspended in 24 mL of medium before being planted in a 6-well plate (Figure 1A). After 2 days, non-adherent cells were removed by completely changing the culture medium. Before changing the medium, a significant number of suspended cells were observed (Figure 1B). After 3 days of culture, small cell colonies began to form. On the sixth day, the size and number of colonie...

Watch this Scientific Journal Video about Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice at JoVE.com

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

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

Economical and Efficient Protocol of Isolating and Generating Bone Marrow-derived Dendritic Cells from Mouse. Procedures involving animal subjects have been approved by the Institution Animal Ethics Committee of Nanjing Medical University.One.Introduction. With the increase in tumor immunity research, the demand for DC cells is gradually increasing.However, DCs are rare in all tissues. The traditional method of mainly inducing the differentiation of bone marrow into DCs is complicated and costly.Two. Isolation of bone marrow and preparation of BM cells We sacrifice mice and fix on mouse operating table.Cut the skin of the left exposed muscles and femoral artery. Do not cut the femoral artery directly. Otherwise, it will cause heavy bleeding and contaminate the field of vision.Cut all the muscles around the femur. Slowly stretch the lower limbs of the mouse outwards until prolapse of the femoral head and can be observed. Separate the lower limbs from the body.Cut the hind leg to obtain a free and complete femur. Remove the muscles using gauze. Do not tear the muscles directly.The femur of mice is fragile. Place the femur in 75%alcohol for two to five minutes.Three. Injection culture of BMDC Rinse the residual alcohol with PBS.Clamp middle and bottom part of the femur with hemostatic forceps, and clamp lower end of the femur with another hemostatic forceps. The hemostatic forceps are applied literally to the femur and the femur is broken from epiphyseal line. Use one ml syringe to pierce the bone marrow cavity from the epiphyseal line.Use complete medium two parts flush the bone marrow cavity until the bone becomes white. Collect flushing fluids. Supplementing the complete medium containing GM CSF/IL-4 to 24ml, and seed in a 6-well plate with 4ml per well.Four.Results.After two days, replace all medium containing GM-CSF/IL-4. Half replaced complete medium containing GM-CSF/IL-4, on the fourth and sixth and eighth day. The number of cells reached a peak on the seventh day and then decreased gradually.DC cells formed a large number of synapsis showing a typical mature DC cell morphology. Flow sub metric analyzes show that the ratio of CD11c imposed what 71%on the sixth day, wide ratio increase to 96.1%on the 10th day. The expression of CD11c, CD80 and MSC2, gradually increased with increasing cultivation time.Five.Conclusion.In short, we established an economical and efficient protocol for isolating and generating bone-marrow-derived dendritic cells from mice, which only takes 10 minutes to separate bone marrow cells. A high number of high purity DCs was harvested after six to seven days of culture with 10ng per ml, GM-CSF, and IL-4.

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