This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Health (NIH), USA.
All mice were maintained at an American Association for the Accreditation of Laboratory Animal Care&#x2013;accredited animal facility at the National Institute of Allergy and Infectious Diseases (NIAID) and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under the auspices of a protocol approved by the Animal Care and Use Committee of the NIAID.

1. Isolation of Mouse Bone Marrow Cells
<ol>
	<li>Euthanize mice using the institution's animal care committee-approved protocol and spray the animal surface with 70% ethanol.</li>
	<li>Make an incision of the skin in the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.</li>
	<li>Cut off the muscles from the lower extremities using scissors and carefully dislocate the acetabulum from the hip joint, while avoiding breaking the femur head.</li>
	<li>Remove the remaining muscles from the femur and tibia using a scalpel and scissors and separate the femur from the tibia at the knee joint exercising care to not break the bone ends. Place the bones in a Petri dish containing ice-cold RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin.</li>
	<li>Proceed to the following steps under a tissue culture hood. Take extra precaution to maintain strict sterile techniques to avoid neutrophil activation.</li>
	<li>Rinse each bone with 70% ethanol (within a Petri dish) followed by three subsequent washes in ice-cold sterile PBS (within Petri dishes) to rinse off the ethanol from the surface of the bones.</li>
	<li>Inside a clean sterile Petri dish, cut off the epiphyses of the bones and keep them aside.</li>
	<li>Use a 25-gauge needle and a 12 cc syringe filled with RPMI supplemented with 10% FBS and 2 mM EDTA, and flush the bone marrow cells from both ends of the bone shafts onto a 50 ml screw top Falcon tube fitted with a 100 &mu;m filter. In order to efficiently remove all cells, scrape the inner surface of the bones using the 25-gauge needle.</li>
</ol>
NOTE: Blanching of bones indicates that the cells have been sufficiently scraped.
NOTE: Use approximately 10 ml of media to flush a femur/tibia pair. Adding EDTA to the medium is essential to prevent clumping of the cells.
<ol start="9">
	<li>Cut the bone epiphyses in small 0.5-1 mm3 pieces with a scalpel and smash them through the 100 &mu;m filter using the back end of a 2.5 ml Eppendorf Combitip Plus Biopur pipette tip.</li>
	<li>Centrifuge at 1,400 rpm for 7 min at 4 &deg;C.</li>
	<li>Lyse the red blood cells by resuspending the cell pellet in 20 ml of 0.2% NaCl for approximately 20 sec followed by addition of 20 ml of 1.6% NaCl. (Critical: Do not exceed 20-30 sec of hypotonic lysis to avoid bone marrow cell death. The use of hypotonic NaCl for lysis is recommended over ACK lysing buffer because the latter has the potential to activate the neutrophils).</li>
	<li>Centrifuge for 7 min at 1,400 rpm at 4 &deg;C to collect the cells.</li>
	<li>Wash cells with RPMI 1640 1X supplemented with 10% FBS and 2 mM EDTA and centrifuge as in step 1.12.</li>
	<li>The yield of bone marrow cells using this method is approximately 60-80 million per uninfected 8-12 week-old C57BL/6 mouse.</li>
</ol>
2. Separation of Neutrophils by Density Gradient Centrifugation
<ol>
	<li>Count the bone marrow cells and resuspend in 1 ml of ice-cold sterile PBS.</li>
	<li>Add 3 ml of Histopaque 1119 (density, 1.119 g/ml) in a 15-ml conical tube.</li>
	<li>Overlay 3 ml of Histopaque 1077 (density, 1.077 g/ml) on the 3 ml of Histopaque 1119.</li>
</ol>
NOTE: Histopaque 1119 and Histopaque 1077 should be warmed to 18-26 &deg;C before use.
Critical Step: Prepare gradients immediately before use as preparing the gradient in advance will result in diffusion between the two layers and suboptimal neutrophil purity and recovery.
Critical Step: Overlaying Histopaque 1077 over 1119 needs to be done slowly in order to avoid mixing the two densities, which will preclude cell separation during centrifugation.
<ol start="4">
	<li>Overlay the bone marrow cell suspension on top of the Histopaque 1077.</li>
</ol>
Critical Step: Overlaying the bone marrow cell suspension over Histopaque 1077 needs to be done slowly in order to avoid disturbing the interface between the cells and Histopaque 1077.
NOTE: Resuspending bone marrow cells from an uninfected mouse in 1 ml of PBS yields neutrophil purity of &gt;90%. However, pooling many bone marrow samples compromises neutrophil purity; for example, resuspending 300 x 106 cells in 3 ml PBS reduces neutrophil purity from &gt;90% to ~80%. Therefore, investigators should perform pilot experiments to identify the ideal cell count/volume conditions for their specific experiments
<ol start="5">
	<li>Centrifuge for 30 min at 2,000 rpm at 25 &deg;C without brake.</li>
</ol>
NOTE: Centrifugation of the gradient at room temperature is critical and essential for effective separation of the neutrophils.
<ol start="6">
	<li>Collect the neutrophils at the interface of the Histopaque 1119 and Histopaque 1077 layers.</li>
	<li>Wash the collected neutrophils twice with RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin and centrifuge at 1,400 rpm for 7 min at 4 &deg;C.</li>
	<li>Count the neutrophils and determine their viability.</li>
</ol>
NOTE: Neutrophils are typically &gt;95% viable and &gt;90% pure as determined by FACS analysis. The typical yield of neutrophils from the bone marrow (i.e. 2 femur and 2 tibia bones) of an uninfected 8-12 week-old C57BL/6 mouse is ~6-12 million cells. This number is substantially greater when neutrophils are harvested from bone marrow of infected animals. Hence, ~30-40 million neutrophils/mouse were recovered when Candida-infected mice were used for cell harvesting 6.
3. Labeling of Neutrophils Using CellTracker Dyes
<ol>
	<li>Resuspend the neutrophils at 5 x 106 cells/ml in PBS prewarmed at 37 &deg;C.</li>
	<li>Add a CellTracker dye at a final concentration of 5 &mu;M.</li>
</ol>
NOTE: CellTracker Green (CMFDA (5-Chloromethylfluorescein Diacetate) and CellTracker Orange (CMTMR (5-(and-6)-4-Chloromethyl Benzoyl Amino Tetramethylrhodamine) was used in this protocol to differentially label neutrophils from wild-type and gene-deficient mice.
NOTE: Prepare a stock solution of 10 mM of CellTracker Green and CellTracker Orange, aliquot, and store at -80 &deg;C until the day of the experiment.
<ol start="3">
	<li>Incubate neutrophils with 5 &mu;M of the corresponding CellTracker dye for 10 min at 37 &deg;C in a shaking water bath in the dark.</li>
	<li>Wash cells twice with ice-cold RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin.</li>
</ol>
NOTE: Efficient washing of the cells after the labeling step with the CellTracker dye is essential to avoid dye cross-contamination before mixing differentially-labeled neutrophil populations for downstream competitive repopulation studies.
4. Adoptive Transfer of Neutrophils in Mice and Analysis of Transferred Neutrophils Using Flow Cytometry
<ol>
	<li>Resuspend neutrophils in ice-cold PBS at a concentration of 25 x 106 cells/ml and inject 200 &mu;l of the suspension into the lateral tail vein so that 5 x 106 neutrophils are transferred per mouse. For competitive repopulation neutrophil studies, mix wild-type and gene-deficient neutrophils at a 1:1 ratio and inject a total of 5 x 106 neutrophils per mouse as above.</li>
</ol>
NOTE: At least up to 10 x 106 neutrophils may be adoptively transferred per mouse without obvious immediate toxicity to the animals.
<ol start="2">
	<li>At different times following adoptive transfer (e.g. 1, 2, 3 or 4 hr post-transfer), euthanize mice and harvest blood, and/or bone marrow and/or other target organ(s) of interest.</li>
	<li>Prepare single cell suspensions from these tissues for quantitative and qualitative analysis of adoptively transferred labeled neutrophils using published protocols 6,15.</li>
	<li>Following live/dead viability staining and Fc blockade, label cells with CD45 (clone 30-F11), Ly6G (clone 1A8) and CD11b (clone M1/70) and gate on live CD45+ Ly6G+ CD11b+ neutrophils. Neutrophils in this gate include native neutrophils of the recipient mouse as well as the adoptively transferred labeled neutrophils, which are FITC+ (if labeled with CellTracker Green) or PE+ (if labeled with Cell Tracker Orange).</li>
</ol>
NOTE: Neutrophils may be tracked in blood, bone marrow and kidney of Candida-infected mice for at least 4 hr post-transfer.
NOTE: Fixation with 2% paraformaldehyde in PBS does not adversely affect the mean fluorescence intensity of the neutrophils labeled with CellTracker Green or CellTracker Orange for at least 48 hr.

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The authors declare that they have no competing interests.

Herein we present a reliable, simple, fast and economical protocol for isolation of large numbers of neutrophils from the bone marrow of mice with high purity and viability using a density gradient centrifugation approach. When this protocol is performed correctly, ~6-12 &times; 106 neutrophils can be recovered from an uninfected mouse and as many as ~30-40 &times;106 neutrophils may be isolated from a mouse after infection 6. The isolated neutrophils are 80-95% pure and &gt;95% viable.</...

Neutrophils are the most abundant leukocytes in humans. They are the main cellular component of the innate immune system and act as a first line of defense against invading microorganisms. Patients with acquired neutropenia and primary immunodeficiencies that affect neutrophil numbers and/or function develop life-threatening invasive bacterial and fungal infections, highlighting the importance of these cells in host defense 1. Immune recognition of invading pathogens at the infection site by their cognate pattern-recognition receptors results in the induction of an orchestrated innate immune response, which leads to secretion of chemoattractants that generate a chemotactic gradient capable of recruiting neutrophils from the bloodstream into the inflamed tissue 2. After neutrophils enter the infection site, they become activated, which leads to cytokine and chemokine production, pathogen uptake, and killing via oxidative and non-oxidative mechanisms 3. Besides their well-recognized role in innate immunity, neutrophils have also been recently shown to play important roles as initiators of effective adaptive immune responses 4. On the other hand, apart from their protective roles in immunity, neutrophils may also mediate tissue injury and immunopathology due to excessive accumulation and/or activation at sites of inflammation, as shown in a variety of infectious and autoimmune diseases 5-7.
Despite the indispensable role of neutrophils in mounting effective innate immune responses and their pleiotropic effector functions in several infectious diseases and inflammatory conditions, technical difficulties with handling these cells and lack of reliable experimental protocols has hindered research with neutrophils over the past decades. Therefore, use of reproducible assays for isolation of neutrophils should facilitate further research on neutrophil-mediated immunological functions ex vivo and in vivo. To date, several methods have been described for the isolation of neutrophils such as density gradient centrifugation of human blood and mouse blood or bone marrow 8,9, positive or negative immunomagnetic enrichment of neutrophils from mouse blood or bone marrow 10,11, and harvesting of neutrophils from the peritoneal cavity of mice following intraperitoneal injection of thioglycollate or other inflammatory agents 12. Although neutrophils can be easily isolated in large numbers from human blood, this method is suboptimal in mice due to the limited volume of mouse blood that precludes isolation of sufficient neutrophils for functional studies or adoptive transfer experiments 13. In addition, although the yield of thioglycollate-elicited cells from the peritoneal cavity is greater compared to that of mouse blood, the purity of neutrophils in the inflammatory peritoneal lavage varies between 60-90%, and the isolated neutrophils exhibit an activated phenotype. Thus, the cells collected using this method can only be used for performing functional studies of activated but not of unstimulated neutrophils, as the mouse peritoneal cavity has few neutrophils at the steady state 12. Instead, the bone marrow is a convenient reservoir for harvesting large numbers of either unstimulated or activated neutrophils 11,14, which can then be used for downstream functional studies such as phagocytosis, killing and degranulation, or for adoptive transfer into recipient mice.
Herein we describe a simple and fast (~ 2 hr) protocol, which provides a high yield (~6-12 &times; 106 neutrophils/uninfected mouse, or up to 30-40 &times; 106 neutrophils/infected mouse) of pure (80-95%) neutrophils with &gt;95% viability from the bone marrow. This method uses commercially available Histopaque, which are density gradient cell separation media consisting of Ficoll and sodium diatrizoate, to separate neutrophils from the bone marrow of mice. This method yields significantly larger numbers of neutrophils per mouse compared to blood or peritoneal cavity, it can be used to collect neutrophils from mice both at steady state or after infection, and it is easier to layer compared to the density gradient centrifugation method that uses discontinuous Percoll gradients consisting of 55%/65%/75% Percoll in PBS 9. In addition, the time and resources required to collect pure neutrophils are significantly decreased compared to neutrophil isolation using Fluorescence-Activated Cell Sorting. Also, because this method does not involve an immunomagnetic enrichment step, it is more cost-effective, and it avoids the exposure of cells to the magnetic column and antibodies, thus decreasing the likelihood of neutrophil activation.
In addition to performing functional studies of isolated neutrophils ex vivo and adoptive transfer of cells into recipient mice, this protocol also describes a method for labeling of isolated neutrophils using different CellTracker dyes. Differential labeling of neutrophils from mice of various genetic backgrounds can be adapted in competitive repopulation studies for tracking the transferred neutrophils in tissues of recipient mice using flow cytometry, which can provide mechanistic insight on the direct role of specific genes in trafficking of neutrophils from the blood into target inflamed organs 6.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>RPMI 1640 1X with L-glutamine and 25 mM HEPES</td>
 <td>Cellgro</td>
 <td>10-041-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (Heat inactivated)</td>
 <td>GemCell</td>
 <td>100500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td>
 <td>GIBCO</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td>Quality Biological Inc</td>
 <td>351-027-101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.2% and 1.6% sodium chloride</td>
 <td>JT Baker</td>
 <td>3624</td>
 <td>Sodium chloride solutions prepared using distilled water and sterile filtered</td>
 </tr>
 <tr>
 <td>Sterile filtered Histopaque 1077</td>
 <td>Sigma</td>
 <td>10771</td>
 <td>Histopaque 1077 needs to be brought to 18-26 &deg;C before use</td>
 </tr>
 <tr>
 <td>Sterile filtered Histopaque 1119</td>
 <td>Sigma</td>
 <td>11191</td>
 <td>Histopaque 1119 needs to be brought to 18-26 &deg;C before use</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline (PBS) without Calcium and Magnesium</td>
 <td>Cellgro</td>
 <td>210-40-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethyl Alcohol (200 proof)</td>
 <td>The Warner Graham Company</td>
 <td>64-17-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellTracker Green (CMFDA, 5-Chloromethylfluorescein Diacetate)</td>
 <td>Invitrogen</td>
 <td>C-7025</td>
 <td>Make stock solutions of 10 mM in DMSO, aliquot and store at -80 &deg;C</td>
 </tr>
 <tr>
 <td>CellTracker Orange (CMTMR, 5-(and 6)-4-Chloromethyl Benzoyl Amino Tetramethylrhodamine)</td>
 <td>Invitrogen</td>
 <td>C-2927</td>
 <td>Make stock solutions of 10 mM in DMSO, aliquot and store at -80 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-mouse CD45 (Clone 30-F11)</td>
 <td>eBioscience</td>
 <td>170451-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Clone 1A8)</td>
 <td>BD Pharmingen</td>
 <td>551461</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Clone 1A8)</td>
 <td>BD Pharmingen</td>
 <td>560599</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b (Clone M1/70)</td>
 <td>eBioscience</td>
 <td>47-0112-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD16/CD32 (Mouse BD Fc Block)</td>
 <td>BD Pharmingen</td>
 <td>553141</td>
 <td>Use at 1:100 dilution</td>
 </tr>
 <tr>
 <td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
 <td>Molecular Probes</td>
 <td>L-23105</td>
 <td>Use at 1:1000 dilution</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>67-68-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde Solution, 4% in PBS</td>
 <td>USB Corporation</td>
 <td>19943</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table 1. Reagents for isolation, labeling and tracking of adoptively transferred neutrophils.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>Taconic</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes</td>
 <td>Corning</td>
 <td>430053</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml centrifuge tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>25 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229225B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229210B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229205B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pasteur pipettes (3 ml)</td>
 <td>BD Falcon</td>
 <td>357575</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12 ml syringes</td>
 <td>Kendall monoject</td>
 <td>512878</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>25 G x 5/8 in. Needles (precision glide needles)</td>
 <td>BD</td>
 <td>305122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 mm cell strainers</td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bactericidal Petri dishes</td>
 <td>BD Falcon</td>
 <td>351029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Combitips Plus Biopur</td>
 <td>Eppendorf</td>
 <td>2249608-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse dissecting instruments (Scissors, forceps, scalpel)</td>
 <td>Biomedical Research Instruments</td>
 <td>10-2300, 10-2165, 25-1200, 26-1000</td>
 <td>Instruments sterilized prior to use</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tissue culture hood</td>
 <td>The Baker Company</td>
 <td>SG403</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Refrigerated centrifuge</td>
 <td>Thermo Fischer Scientific</td>
 <td>75004521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>37 &deg;C shaking water bath</td>
 <td>Thermo Fischer Scientific</td>
 <td>3166721</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table 2. List of Materials and Equipment used in this protocol.</td>
 </tr>
 </tbody>
</table>

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.

This protocol is optimized for the harvest of bone marrow cells from mice and the subsequent separation of neutrophils from these cells by density gradient centrifugation using commercially available Histopaque cell separation media. Neutrophils isolated using this method can be used for a variety of downstream functional studies ex vivo and for adoptive transfer experiments in recipient mice.
The typical yield of collection of bone marrow cells from both femurs and tibia per uninfect...

Watch this Scientific Journal Video about Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments at JoVE.com

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

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 ...

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Immunology and Infection

61.6K vistas.  National Institute of Allergy and Infectious Diseases, NIH. Se describe un protocolo para el aislamiento y purificación de los neutrófilos de médula ósea de ratón por centrifugación en gradiente de densidad y para el etiquetado de neutrófilos usando colorantes CellTracker. Esto representa un método sencillo, rápido, reproducible y económico para la obtención de un gran número de neutrófilos para los estudios funcionales aguas abajo o experimentos de transferencia adoptivos y seguimiento.

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

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.

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 ...

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.

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 ...

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 ...

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 ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

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 ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

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 and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.