This work supported in part by the NIH under Grant R15 CA137499-01 (F.B.) and a startup fund from Ohio University (F.B.).

1. Dissection of mouse femurs and tibiae and culture of bone marrow cells
<ol>
<li>Sacrifice 2 mice by CO2 asphyxiation and carefully dissect tibias and femurs without cutting the bone ends.</li>
<li>Clean the bones from all the attached tissues by using tissue paper. Be careful not to break the bones.</li>
<li>Sterilize the bones by immersion in 70% ethanol for 10 min in a 35 mm Petri dish. From this moment, work inside a biosafety hood to avoid contamination of the cell cultures.</li>
<li>Recover the bones from the ethanol and let them air dry for 5 min in a Petri dish inside the biosafety cabinet.</li>
<li>Cut the femurs in half, and the tibia by its thinnest tip. Infuse the inside of the bone with 1 ml of RPMI medium (without serum but with antibiotics) using a sterile syringe on a sterile Petri dish.</li> 
<li>The cell suspension is collected and washed 2X in RPMI medium by 10 min centrifugation in a 15 ml centrifuge tube at 1,100 RPM in a refrigerated centrifuge (4&deg;C) with a swinging bucket rotor.</li> 
<li>After the last wash, resuspend the cells in 2 ml of ACK lysis buffer and incubate for 5 min at room temperature in order to eliminate red blood cells.</li>
<li>Add 13 ml of RPMI with 10% FBS, resuspend and wash 2X in this medium with the same settings as described in 1.6.</li> 
<li>Count cells, adjust to 2 x 105 cells/ml with RPMI 10% FBS, and add rm-GM-CSF (20 ng/ml final concentration) 5.</li> 
<li>Add 10 ml of this suspension to a sterile, microbiological quality, 10 cm Petri dish, and culture in a CO2 incubator (37&deg;C, 5% CO2).</li> 
<li>Three days later, add another 10 ml of RPMI 10%FBS with 20 ng/l of rmGM-CSF to each of the prepared plates.</li>
<li>Three days later 10 ml of cell suspension are recovered from each Petri dish, centrifuged as in 1.6, resuspended in the same volume of RPMI 10% FBS with rmGM-CSF and returned to the Petri dish. Cells are cultured for 2 additional days in the CO2 incubator.</li>
</ol>
2. Collection and labeling of DCs
<ol>
<li>After 8 days in culture loosely adherent cells are recovered by washing the Petri dishes with fresh medium. This protocol renders around 2-4 x108 DCs in our hands. Cells can be analyzed for DC phenotype by flow cytometry.</li>
<li>Collected cells are then labeled with the Qtracker 655 Cell Labeling Kit strictly following the manufacturer's instructions.</li>
<li>We got excellent results labeling murine bone marrow-derived DCs with Qdots at a 10 nM concentration. To accomplish this, aliquots of Qtracker Cell Labeling Kit components A and B are mixed in equal volumes, incubated for 5 min at room temperature, and immediately diluted 1/100 in fresh medium with vortexing for 30 s.</li>
<li>To stain 5 x 106 DCs, mix 5 &mu;l of each kit components A and B in an eppendorf tube and incubate for 5 min at room temp. Then, add 1 ml of RPMI 10% FBS incubate, and vortex for 30 second.</li>
<li>Add 0.5 ml of cell suspension containing 5 x106 DCs, and incubate at 37&deg;C for 60 min.</li> 
<li>Then, wash cells 2X in RPMI 10% FBS (1,100 RPM). Cells can be resuspended in RPMI for further culture or in PBS for injection in experimental mice.</li>
</ol>
3. Evaluation of labeled DC
<ol>
<li>Cell labeling can be evaluated by means of fluorescent microscopy. To accomplish this, a drop of cell suspension is added to a microscope slide, covered with a glass coverslip, and evaluated with a fluorescent microscope taking into account that these Qdots have emission and excitation spectra of 565nm and 405&#x2013;525nm respectively (Fig. 3)</li>
<li>At this point, the labeled cells can be used to inject animals, or maturation can be induced by incubating these cells for 48 h (37&deg;C, 5 CO2) in the presence of LPS (100 ng/ml) and TNF- (20 ng/ml). Fluorescent labeling is not affected by DC maturation (Fig. 4).</li>
</ol>
4. Representative Results:
Herewith we described how to prepare murine bone marrow-derived DCs and how labeled them with fluorescent Qdots. Fig. 1 summarizes the procedure to obtain the cell, while Fig. 2 depicts the procedure to label them with Qdots. As shown in Fig. 3, almost all the cells are labeled by this procedure, and this is not affected by DC maturation with inflammatory factors (Fig. 4). Fig. 5 shows that Qdot-stained DCs (Fig.5a) behave similar to non-stained cells when treated with an inflammatory cocktail. Both cell preparations similarly upregulate expression of costimulatory molecules (Fig. 5b); and produce similar amounts of IL-6 (Fig. 5c) and nitric oxide (Fig. 5d) upon maturation stimuli. This agrees with previous published data indicating that Qdot staining does not affect the capability of DC to turn into mature cells capable of eliciting immune responses [6]. These cells can then be used for injecting experimental animals. As shown in Fig. 6, 2 days after intravenous injection of labeled DCs into mice, we were able to detect Qdot-stained cells in the spleen. This is also in agreement with previous published data showing the use of Qdot nanocrystals to evaluate migration of DC in vivo [6] by non-invasive imaging and flow cytometry. 
<img src="/files/ftp_upload/2785/2785fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow chart of DC generation from bone marrow. Herewith we depict the process to obtain bone marrow-derived DC. Both tibias and femurs are dissected and clean from surrounding tissue. Bone tips are preserved and the interior of the bones are flushed with medium. After eliminating red blood cells, bone marrow cells are cultured for 8 days in the presence of GM-CSF to differentiate them into DCs. 
<img src="/files/ftp_upload/2785/2785fig2.jpg" alt="Figure 2"/> 
Figure 2. Flow chart of the labeling procedure. Equal aliquots of Qtracker Cell Labeling Kit components A and B are mixed in an eppendorf tube. Dendritic cells are collected from 8-day bone marrow cultures with GM-CSF and mixed with the labeling dye for 60 min at 37C. Then cells are washed in media to eliminate excess labeling particles. 
<img src="/files/ftp_upload/2785/2785fig3.jpg" alt="Figure 3"/> 
Figure 3. Fluorescent microphotography of DCs immediately after labeling. A drop of labeled cells was deposited on a glass slide and covered by a coverslip. Then, samples were evaluated in a fluorescence microscope and images were acquired through a Micropublisher 5.0 Digital CCD Color Camera (Qimaging, Surrey, BC Canada).
<img src="/files/ftp_upload/2785/2785fig4.jpg" alt="Figure 4"/> 
Figure 4. Fluorescent microphotography of DCs after 48 h maturation in vitro. Labeled DCs were cultured for 48 h with LPS (100 ng/ml) and TNF-&alpha; (20 ng/ml) on glass slides on a CO2 incubator (37&deg;C, 5% CO2). Then, samples were washed with PBS (5 min, 2X), fixed with acetone (15 min, 4&deg;C) and counterstained with DAPI. Cells were evaluated in a fluorescent microscope as above. 
<img src="/files/ftp_upload/2785/2785fig5.jpg" alt="Figure 5"/> 
Figure 5. Biological activity of Qdot stained mature DCs. Labeled DCs matured in vitro as above were analyzed by flow cytometry (A) and (B). (A) Qdot-stained cells give a strong signal in the FL3 (PercP) channel. Shaded histogram: unstained control. (B) Qdot-stained and unstained DCs were additionally stained with specific antibodies against maturation markers CD86 and CD40; and isotype controls. Cells were then analyzed in a FACSort flow cytometer (Becton Dickinson, San Jose, CA). Additionally, IL-6 (C) and nitric oxide (NO) were determined in supernatants of mature Qdot-stained DCs and controls. Levels of IL-6 and nitric oxide were determined by means of ELISA analysis and Griess assay as we have previously described 7, 8. All the data showed in this figure is representative of two or three independent experiments showing similar results. Data was analyzed by ANOVA by using the GraphPad software.
<img src="/files/ftp_upload/2785/2785fig6.jpg" alt="Figure 6"/>
 Figure 6. Detection of labeled DCs in dissected tissues. Labeled mature DCs (1x106 cells) were intravenously injected into C57BL/6 mice. (A) Two days later mice were sacrificed and spleens collected, snap frozen, embedded in OCT, and 8 &mu;M sections prepared using a cryostat. Then, samples were fixed with acetone (15 min, 4&deg;C) and counterstained with DAPI. Cells were evaluated in a fluorescent microscope as above. Fig 6a: 40X magnification, Fig 6b: 100X magnification, Fig. 6C, 400X magnification.

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No conflicts of interest declared.

Murine myeloid) DCs have been extensively used in order to determine the efficacy and improvement of DC-based vaccines; investigate DC:T cell interactions or DC development; and determine their role in various diseases 9-11. Herewith we show how to generate DCs from precursors recovered from bone marrows of tibias and femurs. We recover the bones without cutting the tips, allowing us to sterilize them by submersion in ethanol 70%, thus reducing the probability of contamination. To differentiate DCs from bone marrow cells we only use GM-CSF as previously described 5. Although some protocols also use IL-4, it has been reported that this cytokine is not necessary when working with high levels of GM-CSF 12. Indeed, we have previously demonstrated that these DCs are able to induce immune responses 13. Also, care has to be taken to recover only loosely adherent cells from 8-day cultures by washing the Petri dishes with medium since attached cells show a more monocyte-like phenotype. Here we show the labeling of DCs with fluorescent Qdot particles. This labeling has some advantages respect to other methods. First, the Qdots particles are easily incorporated into the cells. Second, the fluorescent signal is very high and is not altered by DC maturation. Third, the fluorescence is not lost when cells or tissues are fixed with solvents such as acetone, contrary to what happens if GFP is used to tag DCs 14, giving more flexibility at the moment to choose staining protocols. Finally, the high fluorescent signal given by these particles allows visualization of the cells despite tissue auto-fluorescence. As previously described 6, Qdot staining did not affect the maturation capability of these cells. Herewith we show that Qdot-stained DCs behave in a similar way as non-stained DCs, upregulating costimulatory molecules, and producing IL-6 and nitric oxide in response to inflammatory stimuli. Although DCs are cells specialized in triggering immune responses, they have been shown to participate in pathological conditions such as cancer and atherosclerosis 4, 15, 16. They have been also claimed to participate in angiogenic process 17, 18, even suggested as structurally participating in the developing of new vessels 19, 20. Thus, methods that allow for DC tracking in vivo, and determining their geographical localization in different tissues 4, 21, 22 are very valuable.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>C57BL/6 mice</td>
 <td>Jackson laboratories</td>
 <td>&nbsp;</td>
 <td>Females, 6-8 weeks old</td>
 </tr>
 <tr>
 	<td>RPMI</td>
 <td>Invitrogen</td>
 <td>11875-119</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fetal bovine serum, qualified</td>
 <td>Invitrogen</td>
 <td>10437-028</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Antibiotic-antimycotic</td>
 <td>Invitrogen</td>
 <td>15240-096</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>PBS</td>
 <td>Invitrogen</td>
 <td>10010-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ACK Lysing Buffer</td>
 <td>Lonza Walkersville, Inc</td>
 <td>10-548E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Recombinant murine GM-CSF</td>
 <td>PeproTech Inc</td>
 <td>315-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Qtracker 655 Cell Labeling Kit</td>
 <td>Invitrogen</td>
 <td>Q25021MP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Lipopolysaccharide</td>
 <td>Invivogen</td>
 <td>tlrl-eblps</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Recombinant murine TNF alpha</td>
 <td>Peprotech</td>
 <td>315-01A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CD86 antibody</td>
 <td>BD Biosciences</td>
 <td>553691</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CD40 antibody</td>
 <td>BD Biosciences</td>
 <td>553791</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Griess reagent system</td>
 <td>Promega</td>
 <td>G2930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>IL-6 capture antibody</td>
 <td>eBioscience</td>
 <td>13-7061-81</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>IL-6 detection antibody</td>
 <td>eBioscience</td>
 <td>13-7062-81</td>
 <td>&nbsp;</td>
 </tr> 
 </tbody>
</table>

generation and labeling of murine bone marrow-derived dendritic cells with qdot nanocrystals for tracking studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Watch this Scientific Journal Video about Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies at JoVE.com

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

generation-labeling-murine-bone-marrow-derived-dendritic-cells-with

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Immunology and Infection

24.8K ビュー.  Ohio University. 樹状細胞の取り込み抗原およびT細胞に処理された抗原を提示する免疫器官に向かって移動する。 Qdot ®二ナノラベルは、長期的かつ安定的な蛍光シグナルを提供します。これは、蛍光顕微鏡により異なる臓器に樹状細胞を追跡できます。

ビデオ: Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

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.

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 ...