The authors wish to thank Anne Daron and Pierre Louis Loyher for editorial assistance, the Plateforme Imagerie Piti&#233;-Salp&#234;tri&#232;re (PICPS) for assistance with the two-photon microscope and the animal Facility &#8220;NAC&#8221; and Camille Baudesson for mice breeding assistance. The research leading to these results has received funding from the European Community&#8217;s Seventh Framework Programme (FP7/2007-2013) under grant agreement n&#176; 304810 &#8211; RAIDs, and n&#176;241440-Endostem, from Inserm, from Universit&#233; Pierre et Marie Curie &#8220;Emergence&#8221;, from la &#8220;Ligue contre le cancer&#8221;, from &#8220;Association pour la Recherche sur le Cancer&#8221; and from &#8220;Agence Nationale de la Recherche&#8221; Programme Emergence 2012 (ANR-EMMA-050).&#160;P.H. was supported by la &#8220;Ligue contre le cancer&#8221;.&#160;

NOTE: All experiment protocols were approved by the French Animal Experimentation and Ethics Committee and validated by the &#8220;Service Protection et Sant&#233; Animales, Environnement&#8221; with number A-75-2065. Sample sizes are chosen to ensure reproducibility of the experiments, and according to the 3R of animal ethic regulation.

1. Preparation of the Mouse
<ol>
	<li>Anaesthesia
	<ol>
		<li>For short period of imaging (less than 1 hr), anaesthetize the mouse with an intraperitoneal injection of 200 &#181;l of a saline solution containing Ketamine (100 mg/kg) and Xylazine (10 mg/kg).</li>
		<li>Alternatively, for longer period of imaging (up to 4 hr), anaesthetize the mouse with an inhalation of isoflurane 2.5% vaporized in a 70/30 mixture of O2/N2O, through an adapted mask.</li>
		<li>NOTE: This way provides for more stable unconsciousness and avoids serial intraperitoneal injection of drugs.</li>
		<li>Once the mouse is anesthetized, check for unconsciousness through stimulation of the foot pad with nippers.</li>
	</ol>
	</li>
	<li>Vascular staining/ additional staining
	<ol>
		<li>For vascular staining: Inject intravenously in the tail vein, 200 &#181;l of Rhodamin-Dextran (2 MDa, 10 mg/ml in saline buffer) .</li>
		<li>For neutrophil staining: inject 2 &#181;g of Ly6G-PE (clone 1A8) in 100 &#181;l of saline solution intravenously in the tail vein.</li>
	</ol>
	</li>
	<li>Immobilization
	<ol>
		<li>For immobilization, use a custom made stereotactic holder adapted to the microscope and to the anaesthetic gas inhalator. 
		NOTE: The sterotactic holder is a metal support with two metal plate brackets, one being fixed and the other removable and adaptable, to tighten the animal&#8217;s head.</li>
		<li>Install the head of the mouse between the retaining plates to ensure isolation from breathing movements. Tighten the ears between retaining plates and head to stretch the skin. Check for stability and breathing welfare of the animal.</li>
	</ol>
	</li>
	<li>Remove the scalp
	<ol>
		<li>Carefully use ethanol 70% to wet the hair of the scalp with a sterile applicator, avoiding contact with the eyes.</li>
		<li>Cut the skin with sterile scissors and nippers to fully remove the scalp from the backward of the parietal bone to the frontal bone. Keep away up to 3 mm from eyes and ears to prevent extensive bleeding. Remove the loose connective tissue covering the skull (periosteum). Wash out remaining hair with warm PBS-soaked paper towel.</li>
	</ol>
	</li>
	<li>Immersion system set up
	<ol>
		<li>Paste a rubber ring of 18mm diameter with surgical glue directly on the skull using a small gauge needle to control distribution. Glue the entire periphery of the rubber ring to prevent leakage (30 &#181;l should be sufficient).</li>
		<li>Clean the skull under the ring one last time with PBS-soaked paper towel and then add 37 &#176;C PBS for complete immersion of the skull. Add a drop of NaCl 0.9% solution or specific ophthalmic ointment on each eye of the mouse to avoid eye dryness.&#160;&#160;Drag the stereotactic holder under the objective.</li>
		<li>Approximately position the field using the microscope ocular through direct transmission.</li>
	</ol>
	</li>
</ol>
2. Two-photon Imaging Acquisition
<ol>
	<li>Use a multiphoton microscope coupled with a Ti:Sapphire crystal laser, which provides 140fs pulses of NIR light, selectively tunable from 680 to 1,080 nm, and an acousto-optic modulator for laser power control. Use a configuration including 3 external non-descanned detectors (NDD) (that enable simultaneous recording of 3 fluorescent channels) with a combination of 2 dichroic mirrors (565&#160;nm and 690&#160;nm), 565/610 and 500/550 bandpass filters, and a 485 short pass filter, with a plan apochromat &#215;20 (NA = 1) water immersion objective.</li>
	<li>Set the heating chamber to allow good homeothermy of the anaesthetized mouse., 
	NOTE: Temperature might be set 1 hr&#160;before imaging session for better stability. In our case, 32 &#176;C has been chosen to get optimal body temperature of the anaesthetized mouse (36-37 &#176;C) and is maintained up to 4 hr. This temperature may be adapted to the system used (digital probe can be used to check this point). In addition, the heating chamber used is dark/black and covers a part of the system (objectives and motorized plate) to avoid external light contamination.</li>
	<li>Turn on the system
	<ol>
		<li>Turn on the Ti:Sapphire laser, then the whole microscope system. Launch the acquisition software. Set the laser wavelength range to 870 nm. Select appropriate NDD for recording. 
		NOTE: In our case, second harmonic generation (SHG) 19and cyan fluorescent protein (ECFP) are detected by the NDD after the 485 short pass filter. ECFP is also detected by the NDD after the 500/550 bandpass filter and rhodamin-dextran is detected by the NDD after the 565/610 bandpass filter.</li>
	</ol>
	</li>
	<li>Adjust acquisition settings
	<ol>
		<li>Use the &#8220;live acquisition&#8221; imaging command of the software and using the microscope directional control, adjust the focus on a region with ECFP and Rhodamine signal. Set the laser power to the minimum to obtain best signal-to-noise ratio with minimal photo-damage. 
		NOTE: The power setting used varies according to the depth of the region of interest. Typically the AOM settings are set around 20%, which represents a laser power beyond the objective around 15 mW. It is better to increase gain power on the PMTs for each NDD to reduce photo-bleaching and photo-damage. Typical acquisition settings are single scanning with a pixel dwell time of 1.58 &#181;s, and a resolution of 512 x 512 pixels with a 1.5 magnification.</li>
	</ol>
	</li>
	<li>Select regions of interest
	<ol>
		<li>Once recording parameters are set, use again the &#8220;live&#8221; acquisition command, and survey the tissue to choose a desired (x,y,z) field to be monitored in real time. 
		NOTE: Typically, we chose a field including both vascular and parenchymal areas.</li>
		<li>Register the field with the &#8220;position&#8221; software command. 
		NOTE: This command memorizes x,y,z coordinates of different distant fields.</li>
		<li>Select and register additional fields of interest (up to 3) using the same procedure.</li>
		<li>Set a 3D z-stack with the &#8220;z-stack&#8221; software command on the first memorized position according to the required volume with chosen magnification. Memorize deeper position first, and then the highest position. Normalize laser power with the depth. 
		NOTE: For multiple concomitant field imaging, we acquire 5 slices separated by 3 &#181;m z-steps for each field to acquire a volume of 12 &#181;m thickness. These settings might be adapted to the type of cell studied.</li>
	</ol>
	</li>
	<li>Launch acquisition
	<ol>
		<li>Select the &#8220;time series&#8221; software command and choose the duration of time interval and the number of cycles. Begin time-lapse imaging according to the time lapse and duration required by selecting the &#8220;start experiment&#8221; software command. 
		NOTE: In our case, we acquire one volume each 30 sec&#160;during 60 cycles representing 30 min real time. Smaller time lapse can be performed to track fast cell rolling in the blood vessel. In this case thinner z-stack and no more than 2 different fields can be recorded at the same time.</li>
		<li>Regular monitoring of the set up.
		<ol>
			<li>Always monitor the animal to make sure it is unconscious. 
			NOTE: Shaking of whiskers or legs of the animal are indicators of revival. In case the animal&#8217;s breathing is jerky, the quantity of isoflurane can be slightly reduced. Strong tissue drift during acquisition is an indicator of mouse revival.</li>
			<li>Ensure the objective is properly immersed. Renew 37 &#176;C PBS between two acquisitions (30 min). 
			NOTE: Progressive disappearance of signal during acquisition is indicator of PBS leakage.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>End of procedure
	<ol>
		<li>Once the required videos are recorded, euthanize the animal by cervical dislocation rapidly before resuscitation. 
		NOTE: wound healing leads to the formation of a scab in 24 to 48 hr which limits the possibility to perform longitudinal imaging.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>Drift correction
	<ol>
		<li>Use Imaris Bitplane software for 3D automatic tracking.</li>
		<li>Open the 3D image sequence on Imaris. Select &#8220;spot&#8221; command, and generate manual tracking (mouse click+shift) for all time points for a minimum of 4 different anchor points, if possible homogeneously distributed in the field.</li>
		<li>Apply the drift correction using &#8220;correct drift&#8221; command in the &#8220;edit track&#8221; tab.</li>
		<li>Check for the quality of the correction in x, y and particularly in z to avoid artefactual wavering. 
		NOTE: If the correction is not satisfactory, try other anchor points or exclude the video from the analysis.</li>
	</ol>
	</li>
	<li>Cell tracking
	<ol>
		<li>Use &#8220;add new spots&#8221; command and select &#8220;track spots over time&#8221; algorithm setting. Go to next step.</li>
		<li>Apply automatic cell tracking procedure either on the whole channel of interest using &#8220;source channel&#8221; command. Set objects (i.e., cells) diameter to 10 &#181;m. Go to next step. 
		NOTE: The choice will depend on the distinction between objects and specificity of the measured signal. In short, automatic tracking on the whole channel is possible only if detected signal is specific to the object of interest. Because both SHG and ECFP are detected by the NDD after the 485 short pass filter, tracking will be more specific and efficient using the ECFP signal recorded by the NDD after the 500/550 bandpass filters. Clustered or dense packed objects will require manual selection of individual objects similar to step 3.1.2.</li>
		<li>Select &#8220;quality&#8221; filter type, set the threshold to exclude tracking of unspecific objects. Go to next step.</li>
		<li>Choose &#8220;Brownian Algorithm Motion&#8221; with accurate parameters of &#8220;Max distance&#8221; and &#8220;Gap size&#8221; between two spots. Validate track generation.</li>
		<li>Once tracks are automatically calculated, select &#8220;track duration&#8221; filter type, set the threshold to eliminate tracks shorter than 120 sec (representing 4 time points).</li>
		<li>Mandatory: After automatic tracking, check the quality of the tracks.
		<ol>
			<li>Unroll the time bar to check that the objects follow the track paths. If not, use the options of track point correction available in the &#8220;edit&#8221; tab: Delete false or improper tracks with &#8220;delete&#8221; command (delete any track point individually using &#8220;disconnect&#8221; command). If track path is discontinuous, add missing time points (mouse click+shift), select all points on a given track, and use &#8220;connect&#8221; command in the &#8220;edit track&#8221; tab.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Quantification parameters
	<ol>
		<li>Select the &#8220;Statistic&#8221; tab in &#8220;Preferences&#8221; command, choose the dynamic parameters of interest in the list. Select &#8220;Export all statistics to file&#8221; command and save the excel file generated. 
		NOTE: Several dynamic parameters are directly obtained from the software such as track lengths, instantaneous velocity, mean velocity, and track straightness. Motility coefficient 6,20 can be determined but is not directly provided by the software.</li>
		<li>For each cell, calculate the mean of the displacements for each period of time (&#948;t) (for a 30 sec time lapse movie, &#948;t will be 30, 60, 90 sec, etc). Plot the mean of the square displacement as a function of the time interval. Obtain motility coefficient by calculating the slope of the linear part of the curve.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The critical points of in vivo imaging methodology are to ensure stability of the focus in order to maximize the duration of imaging and to minimize the risk of bacterial contamination and inflammation, which might impact the dynamics of the inflammatory cells. Imaging of the skull bone marrow follows these aims as the surgery performed to get access to the bone marrow is minimal. The use of sterile material and antiseptics is essential to limit the risk of infection that might induce perturbation in cel...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

<table><tbody><tr><td>Ketamin</td><td>Merial</td><td></td><td>100 mg/ml, anesthetic</td></tr><tr><td>Xylazin</td><td>Bayer HealthCare</td><td></td><td>10 mg/ml, anesthetic</td></tr><tr><td>Isofluran</td><td>Baxter</td><td></td><td>2.5%, anesthetic</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/NO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>70/30 mixture, anesthetic</td></tr><tr><td>Rhodamin-Dextran</td><td>Invitrogen</td><td></td><td>2 MDa, 10 mg/ml, Vascular staining</td></tr><tr><td>Ly6G-PE</td><td>Becton-Dickinson</td><td></td><td>clone 1A8, neutrophils staining</td></tr><tr><td>Stereotactic holder</td><td>Home made</td><td></td><td>surgery</td></tr><tr><td>Ethanol 70%</td><td></td><td></td><td>surgery</td></tr><tr><td>Sterile scissors and nippers</td><td></td><td></td><td>surgery</td></tr><tr><td>Rubber ring</td><td></td><td></td><td>18 mm diameter, surgery</td></tr><tr><td>Glubran 2</td><td>Queryo Medical</td><td></td><td>Surgical Glue, rubber ring fixation</td></tr><tr><td>Small gauge needles</td><td>Terumo</td><td></td><td>surgery</td></tr><tr><td>Zeiss LSM 710 NLO multiphoton microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>Microscope</td></tr><tr><td>Ti:Sapphire crystal laser&amp;nbsp;</td><td>Coherent Chameleon Ultra</td><td></td><td>140 fsec pulses of NIR light</td></tr><tr><td>Zen 2010</td><td>Carl Zeiss</td><td></td><td>Acquistion Software</td></tr><tr><td>Imaris Bitplane&amp;nbsp;</td><td>Bitplane</td><td></td><td>Analysis Software, 3D automatic tracking</td></tr><tr><td>PBS 1X</td><td>D. Dutscher</td><td></td><td>surgery</td></tr><tr><td>Thermostated chamber</td><td>Carl Zeiss</td><td></td><td>intravital imaging</td></tr></tbody></table>

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.

Mouse skull structure offers a good opportunity to study the bone marrow physiology by intravital imaging. The bone being thin around the front-parietal area, it is possible to get access to medullar niches without abrasion of the bone. Figure 1 represents a wide 2D field of the skull of a MacBlue transgenic mouse. The bone matrix is mainly composed of collagen I easily detectable by SHG 19. Injection of rhodamin-dextran stains the vascular network of the bone marrow and allows for the identif...

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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.

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 ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

9.5K Views.  Sorbonne Universités, UPMC Univ Paris 06, CR7. 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. 

Video: Tracking Mouse Bone Marrow Monocytes In Vivo

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 ...

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Medicine

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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.

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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Biology

Intrafemoral Injection for Bone Marrow Study in Live Mice

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Bone Marrow Harvest from Mouse Hind Limb

Source: Zhu, Y. P., Padgett, et al. <a href="https://www.jove.com/t/59617">Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells.</a> J. Vis. Exp. (2019).
This video describes a detailed protocol to isolate fresh whole bone marrow from the mouse&#39;s hind limbs to investigate whole BM defects for patients with blood disorders such as leukemia.

Source: Zhu, Y. P., Padgett, et al. Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells. J. Vis. Exp. (2019).
This ...

Tracking Mouse Bone Marrow Monocytes In Vivo

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