This work was funded by Pilot and Feasibility Study grant from UCLA-CURE Center, the Crohn's and Colitis Foundation of America Career Development Award (#2691) and National Institute of Health NIDDK K01 (DK084256) funding to Hon Wai Koon.
Bone marrow irradiation operation was assisted by Bernard Levin and Scott Kitchen of UCLA Center for AIDS Research Mouse/Human Chimera Core facility. Flow cytometry operation was assisted by UCLA Vector Core facility.

1. Before-you-start Technical Considerations
<ol>
	<li>We recommend using both C57/BL6 wild-type mice and knockout mice for experiment because mice with corresponding CD molecules can be purchased from major mouse vendors.</li>
	<li>Since it is difficult to track the wild-type mice and knockout mice derived donor cells after bone marrow transplantation, it is necessary to use CD45.1 mice to represent wild-type mice. (CD45.1 C57/SJL WT mice Jackson Laboratories stock #002014).</li>
	<li>Most mouse colonies are CD45.2. Our knockout mice belong to CD45.2 genotype (alternatively a CD45.2 WT mice source: Jackson Laboratories stock #000664). The determination of CD45.1 or CD45.2 genotype can be performed using flow cytometry of bone marrow and peripheral blood as mentioned in section 7 in this protocol.</li>
	<li>Mice after irradiation have compromised immunity. Keep mice in pathogen free facility.</li>
	<li>Operation of irradiator requires special security clearance and training. To save time in training and approval processes, it is best to collaborate with a laboratory with qualified technician who is able to operate the irradiator.</li>
	<li>Many institutions have flow cytometry laboratories with experienced technicians. To minimize problems with flow cytometry operation, it is better to discuss your flow cytometry experiment plan with the experienced technician before you start.</li>
	<li>Mice were assigned in this manner (10 mice per group):</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Group</td>
			<td>&nbsp;</td>
			<td>Bone marrow donor to recipient</td>
			<td>Treatment</td>
		</tr>
		<tr>
			<td>A</td>
			<td>BM exchange</td>
			<td>CD45.1 WT to CD45.2 KO</td>
			<td>DSS colitis</td>
		</tr>
		<tr>
			<td>B</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Water normal control</td>
		</tr>
		<tr>
			<td>C</td>
			<td>BM exchange</td>
			<td>CD45.2 KO to CD45.1 WT</td>
			<td>DSS colitis</td>
		</tr>
		<tr>
			<td>D</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Water normal control</td>
		</tr>
		<tr>
			<td>E</td>
			<td>Sham</td>
			<td>CD45.1 WT to CD45.1 WT</td>
			<td>DSS colitis</td>
		</tr>
		<tr>
			<td>F</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Water normal control</td>
		</tr>
		<tr>
			<td>G</td>
			<td>Sham</td>
			<td>CD45.2 KO to CD45.2 KO</td>
			<td>DSS colitis</td>
		</tr>
		<tr>
			<td>H</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Water normal control</td>
		</tr>
	</tbody>
</table>
* A brief illustration of experimental protocol is shown in Figure 1.
2. Recipient Mice Irradiation
<ol>
	<li>Mice are bred and kept in pathogen-free facility. Place mice in autoclaved irradiation pie with filter. Place one mouse in each slot of irradiation pie. Place the pie into the irradiator and make sure the turntable and pie are turning.</li>
	<li>Turn on the air-pump for ventilation. Close the irradiator door and lock it.</li>
	<li>Irradiate the mice with 1,000 rad (which is equivalent to 10 Gy) around 10 min (depending on radiation source and half-life). From this time point, the irradiated mice are immuno-compromised and have weak immunity against infection. After irradiation, take the pie out and place in a sterile container for transportation to biosafety cabinet in animal facility. Avoid exposing the mice to external environment to minimize chance of infection.</li>
	<li>In biosafety cabinet, transfer the mice into autoclaved mouse cages with autoclaved bedding (4 mice per cage. Add new sterile water with sulfatrim suspension - 3.12 ml per 100 ml water).</li>
	<li>Wrap the water bottle with aluminum foil as the antibiotics are light sensitive. Shake to mix the sulfatrim solution bottle well before loading it to the cage.</li>
</ol>
3. Donor Bone Marrow Extraction
<ol>
	<li>Sacrifice mice with carbon dioxide or isoflurance and then immerse in 1:200 Lysol solution. Spray the mice with 70% ethanol to wet the skin and dissect open bones.</li>
	<li>Soak the dissection instruments in 70% ethanol for sterilization.</li>
	<li>Perform the dissection in a biosafety cabinet. Use the sterile dissection instruments to expose the humerus, femur, tibia and fibula bones, free from attaching muscles and ligaments.</li>
	<li>Cut open both ends of bones to show the red bone marrow. Hold the bones with forceps. Flush the red bone marrow out with cold PBS with 3 ml syringe and 25G needles to a Petri dish. Flush from both ends of bones to yield more bone marrow cells.</li>
	<li>Use 6 ml syringes and 18G1/2 needles to break red bone marrow plugs by repeated aspiration and ejection. Transfer bone marrow with 50 ml Falcon tubes.</li>
	<li>Spin the bone marrow down in 1,800 rpm for 5 min at 4 &deg;C. Discard supernatant, resuspend pellet by vortexing for 5-10 sec.</li>
	<li>Dilute 10X RBC lysis buffer with sterile water. Add 1X RBC lysis buffer (5 ml per donor mouse), vortex again for 5 sec and keep exactly 3 min at room temperature.</li>
	<li>After 3 min, fill the tubes with 20 ml cold PBS to stop the lysis and mix. Pour the content through a 40 &mu;m cell strainer directly into a new 50 ml Falcon tube.</li>
	<li>Rinse the original tubes with 5 ml of PBS and transfer to cell strainer as well. Top up to 50 ml with PBS and mix.</li>
</ol>
4. Counting Bone Marrow Donor Cells
<ol>
	<li>Add 100 &mu;l of Trypan blue to an Eppendorf tube and 100 &mu;l of bone marrow suspension to an Eppendorf tube and mix. Pipette the stained cell mixture to a hemocytometer.</li>
	<li>The amount of cells is calculated by total living bone marrow cell count in the Falcon tubes = unstained cell number in 16 squares x 2 (due to Trypan blue dilution) x 10,000 x 50 ml</li>
	<li>Spin down the bone marrow cells in 50 ml Falcon tubes at 2,000 rpm for 5 min at 4 &deg;C.</li>
	<li>Remove the supernatant. Based on the total cell counts, resuspend the pellet with PBS to 1 x 108 cells per ml</li>
</ol>
5. Infusion to Recipient Mice
<ol>
	<li>Warm the recipient mice on a heat pad and under a warming lamp. Put the recipient mice in restrainer. Inject 1 x 107 cells per mouse in 100-200 &mu;l intravenously.</li>
	<li>Injection of donor bone marrow must be done between 4-24 hr after irradiation.</li>
	<li>Keep up to 4 injected mice per cage. Maintain the injected recipient mice in autoclaved cages with antibiotics-treated water for the first 4 weeks in pathogen free animal facility and let the mice regain immunity. Change cages, antibiotic-treated water and food every 4 days to maintain hygiene.</li>
</ol>
6. Induction of Colitis and Evaluation of Colitis
<ol>
	<li>4 weeks after irradiation and bone marrow transplantation, switch to regular water without antibiotics and maintain the mice for 2 more weeks to regain their normal gut microflora.</li>
	<li>6 weeks after bone marrow injection, measure initial body weight. Some mice are given 5% dextran sulfate in drinking water ad libitum to induce colitis. Some mice are given regular drinking water only as normal control groups.</li>
	<li>5 days later, withdraw peripheral blood, transfer to heparin coated tubes (Vacutainer) and keep in ice. Pool the blood from 4 mice to 1 tube. Combine 300 &mu;l of blood with 300 &mu;l of cell staining buffer = 600 &mu;l. Divide the blood to 5 Eppendorf tubes, each with 100 &mu;l.</li>
	<li>Sacrifice the mice with carbon dioxide or isoflurane. Evaluate macroscopic damage scores (please refer to another JOVE video publication for details 1). Measure body weight change. Measure bowel length and thickness with digital caliper. Observe stool texture (hard, soft or bloody). Determine occult blood in stool with Hemooccult. Dissect colon tissues and fix in formalin for H&amp;E staining.</li>
	<li>Dissect 1 femur bone per mouse, extract bone marrow and flush the bone marrow with cold PBS via 25G needle and 1 ml syringe. Pool 4 bone marrow plugs to 1 tube and keep in ice.</li>
	<li>Pour the bone marrow to Petri dish, shear the bone marrow plug with 18G needle and 5 ml syringe several times until no bone marrow plug is present.</li>
	<li>Spin down with 1,500 rpm for 5 min at 4 &deg;C. Remove supernatant and resuspend with 600 &mu;l cell staining buffer. Divide the resuspended bone marrow to 5 Eppendorf tubes, each with 100 &mu;l.</li>
</ol>
7. Quality Inspection of Bone Marrow Transplantation by Flow Cytometry
<ol>
	<li>Label each sample tubes of blood or bone marrow #1-5:</li>
	<li>Prepare the antibody mixture for each respective tube in the dark.</li>
</ol>
#1 No antibody 
#2 30 &mu;l FITC isotype control + 30 &mu;l PE isotype control + 15 &mu;l CD16/32 blocking 
#3 30 &mu;l FITC CD45.1 Ab + 15 &mu;l CD16/32 blocking 
#4 30 &mu;l PE CD45.2 Ab + 15 &mu;l CD16/32 blocking 
#5 30 &mu;l FITC CD45.1 Ab + 30 &mu;l PE CD45.2 Ab + 15 &mu;l CD16/32 blocking
<table border="1">
	<tbody>
		<tr>
			<td>Add nothing to blood or bone marrow sample #1</td>
		</tr>
		<tr>
			<td>Add 5 &mu;l of antibody mixture #2 to each blood or bone marrow sample #2</td>
		</tr>
		<tr>
			<td>Add 3 &mu;l of antibody mixture #3 to each blood or bone marrow sample #3</td>
		</tr>
		<tr>
			<td>Add 3 &mu;l of antibody mixture #4 to each blood or bone marrow sample #4</td>
		</tr>
		<tr>
			<td>Add 5 &mu;l of antibody mixture #5 to each blood or bone marrow sample #5</td>
		</tr>
	</tbody>
</table>
<ol start="3">
	<li>Keep in dark in ice for 30 min.</li>
	<li>Add 2 ml 1X RBC lysis buffer to each tube. Keep in dark on ice for 15 min.</li>
	<li>Spin down the cells at 1,500 rpm, 5 min at 4 &deg;C. (GH 3.8 rotor, 1,500 rpm = 350 x g) Remove supernatant. Resuspend the pellet with 500 &mu;l cell staining buffer in the dark, then vortex briefly.</li>
	<li>Check the CD45.1 (representing WT) and CD45.2 (representing KO) in the immunostained blood and bone marrow samples by flow cytometry. Select FITC to represent WT CD45.1 and PE to represent KO CD45.2.</li>
	<li>Analyze the flow cytometry results by FlowJo software. Calculate the ratio of FITC:PE ratio or PE:FITC ratio to determine whether donor genotype is dominant genotype in blood and bone marrow.</li>
</ol>

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Liver Physiol. 296, 1167-1179 (2009).</li><li>Castagliuolo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+neurokinin-1+receptor+during+colitis+in+mice:+role+of+the+epidermal+growth+factor+receptor.">Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.</a> Br. J. Pharmacol. 136, 271-279 (2002).</li><li>Koon, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathelicidin+signaling+via+the+Toll-like+receptor+protects+against+colitis+in+mice.">Cathelicidin signaling via the Toll-like receptor protects against colitis in mice.</a> Gastroenterology. 141, 1852-1863 (2011).</li><li>Lee, C. 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No conflicts of interest declared.

This bone marrow transplantation approach is suitable for immunology research of colitis, infection, cancer, obesity and other diseases. This bone marrow transplantation experiment is needed when the gene-of-interest is expressed in both bone marrow derived and non-bone marrow derived cells and the gene-of-interest is suspected to mediate disease by cells from either population. For example, antimicrobial peptide cathelicidin is shown to modulate acute colitis. But it is expressed in both epithelial cells and immune cell...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Cell staining buffer</td>
			<td>Biolegend</td>
			<td>#420201</td>
		</tr>
		<tr>
			<td>10X RBC lysis buffer</td>
			<td>Biolegend</td>
			<td>#420301</td>
		</tr>
		<tr>
			<td>FITC mouse isotype control</td>
			<td>Biolegend</td>
			<td>#400207</td>
		</tr>
		<tr>
			<td>PE mouse isotype control</td>
			<td>Biolegend</td>
			<td>#400211</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD45.2</td>
			<td>Biolegend</td>
			<td>#109807</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.1</td>
			<td>Biolegend</td>
			<td>#110705</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32 blocking</td>
			<td>Biolegend</td>
			<td>#101301</td>
		</tr>
		<tr>
			<td>40 &mu;m cell strainer</td>
			<td>Fisherbrand</td>
			<td>#22363547</td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>MP biomedicals</td>
			<td>#1860454</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisherbrand</td>
			<td>#BP2425</td>
		</tr>
		<tr>
			<td>Sulfatrim (SMZ)</td>
			<td>Qualitest</td>
			<td>NC9242720 (fisher)</td>
		</tr>
		<tr>
			<td>Lysol IC</td>
			<td>Andwin Scientific</td>
			<td>NC9745686 (fisher)</td>
		</tr>
		<tr>
			<td>Vaccutainer</td>
			<td>BD</td>
			<td>#8000813</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Braintree Scientific</td>
			<td>#TV-150</td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>J.L. Shepherd and Associates</td>
			<td>Mark I 68A</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>BD FACSCanto II</td>
		</tr>
		<tr>
			<td>Flow cytometer test tube</td>
			<td>Falcon</td>
			<td>#352052</td>
		</tr>
		<tr>
			<td>Digital caliper</td>
			<td>Fisherbrand</td>
			<td>14-648-17</td>
		</tr>
		<tr>
			<td>Hemoccult ICT</td>
			<td>Beckman Coulter</td>
			<td>G0328QW</td>
		</tr>
	</tbody>
</table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

If the gene-of-interest plays a significant role in immune cells during development of colitis, the mice receiving bone marrow of different genotype through bone marrow transplantation (WT to KO or KO to WT) should have an altered response to DSS colitis. One of the most important parameters for determining the severity of colitis is the H&amp;E staining of the colonic tissues. Colonic tissue structure changes and signs of inflammation can be quantitatively evaluated by H&amp;E histology scoring system. Criteria for H&am...

Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

differentiating-functional-roles-gene-expression-from-immune-non

Research

JoVE Journal

Immunology and Infection

17.4K 次观看.  The University of California Los Angeles, Los Angeles. 骨髓移植提供了一种方法来改变的骨髓衍生细胞的基因型。如果感兴趣的基因的表达在两个骨髓来源的细胞和非骨髓来源的细胞，骨髓移植可以改变到一个不同的基因型的骨髓来源的细胞，不改变非骨髓来源的细胞的基因型。

视频: Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...