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 cell homeostasis.
The development of the stereotactic holder might be challenging and requires specific customization for each system (distance between objective and motorized stage of the microscope, use of gas inhalator for anaesthesia). It is important to get the head of the mouse as horizontal as possible for a wider imaging surface access. Once the animal is positioned, it is likely that several minutes can be required to obtain good stability of the focal plane. Therefore, it is recommended to check for tissue drift before launching the acquisition recording. Because this process together with the selection of regions of interest can last a while, it is also recommended to regularly check that the animal is unconscious if Ketamin /Xylazin have been used, and to check for proper immersion of the objective as leakage and evaporation can occur.
The good positioning of the rubber ring on the skull represents another critical step in the process. Cyanoacrylate glue provides good results in terms of stability and sealing, but one needs to prevent excessive loading of glue, which could cover the skull and block access to the region of interest of the tissue. The advantage of direct immersion of the skull without a glass coverslip is to get access to deeper regions of the bone. In addition, since the skull is not a plain surface, this will not limit the imaging area to the contact surface between the skull and the coverslip, allowing a wide imaging field, as displayed in Figure 1.
For all imaging technologies, the choice between acquisition speed and resolution quality is a compromise. Thus, the number and the quality of images per unit of time are limited, and the choice depends on the scientific question addressed. Typically, vascular monocytes are fast rolling cells, whereas parenchymal monocytes are slow motile or sessile. An accurate tracking of rolling cells requires higher time resolution of acquisition, as shown in Figure 2. High imaging rate for sessile cells is useless and high x,y,z resolution can be preferred to analyze protrusive activity of dendrites for example. Fast cells would require thicker volume to get a longer tracking of their displacement in z.
The mean square displacement as a function of time is not provided by the software Imaris. The principle of this representation based on the fact that, for an object travelling ballistically without any encounter or structural constriction, the distance it travelled would be proportional to the time interval (Figure 2D). Thus linearity of the slope indicates a random walk in a non-constrained environment. In contrast, asymptotic shape of the curve would reflect a constrained displacement over time of the cell population. The second advantage of this calculation is that velocity is sometimes overestimated by artefactual displacement of the centre of mass. This is particularly important for slow motile cell with protrusive activity. Thus, calculation of the motility coefficient by the slope of the curve calculated by linear regression indicates a real displacement of the cellover time 21.
Ly6G staining in vivo illustrates the possibility to add a new parameter of analysis during the imaging process (Figure 3). We have used this technique to stain vascular neutrophil infiltration into skin wound, and observed no defect in their mobility suggesting no functional alteration (Rodero et al in press). Although more than 98% of vascular neutrophils are properly labeled, the staining of bone marrow neutrophils is less efficient, and the mean fluorescence intensity is strongly reduced, suggesting reduced access of the antibody toward the bone marrow parenchyma (data not shown). Rhodamin dextran, quantum dots, or other specific dyes of apoptotic or necrotic cells such as Dapi, Propidium iodide, sytox 22, or fluorescent reporter for cell functions such as production of reactive oxygen species 23, can be added intravenously through the tail vein during imaging process and offer a good opportunity to quantify functional properties such as cell death, phagocytosis and neutrophil extracellular trap. Mazo et al12 nicely used two different vascular dyes with low and high molecular weight to better contrast parenchyma and vasculature. The choice of the fluorescent dye is critical to allow concomitant acquisition of the different fluorescent reporters. Indeed, the excitation wavelength will be chosen accordingly to get the best compromise between all used fluorescent dyes.
The protocol used herein makes it possible to analyze in vivo the biological activity of a cell compartment, study of which was difficult so far. Histological analysis of the bone marrow tissue usually requires a long preparation of bone decalcification to allow thin section, and it only provides a static view of the tissue. The dynamic view highlights the rate of cell exchange between the parenchyma and the bone marrow sinusoids. This rapid process is a crucial step to decipher in order to have a better understanding of cell mobilization mechanism upon inflammatory signal 5 or preconditioning myeloaplasive chemotherapy regimen 6. We have reported event of cell intravasation6 but this process is barely detectable due to the low frequency at steady state. However, it is likely to be enhanced upon inflammatory signaling. Chemokine receptors such as CCR2, CX3CR1 or CXCR4 are highly implicated in the process of intravasation, hence the use of genetic invalidation of these receptors should provide novel fundamental insights in the pathways of cell migration

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.

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			<td>10mg/mL, anesthetic</td>
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			<td>70/30 mixture, anesthetic</td>
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			<td height="24" style="height:24px;width:297px;">Rhodamin-Dextran</td>
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			<td>2MDa, 10mg/mL, Vascular staining</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:297px;">Ly6G-PE</td>
			<td style="width:171px;">Becton-Dickinson</td>
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			<td>clone 1A8, neutrophils staining</td>
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			<td height="21" style="height:21px;width:297px;">Stereotactic holder</td>
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			<td>18mm diameter, surgery</td>
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			<td height="21" style="height:21px;width:297px;">Glubran 2</td>
			<td style="width:171px;">Queryo Medical</td>
			<td style="width:119px;"></td>
			<td>Surgical Glue, rubber ring fixation</td>
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			<td height="21" style="height:21px;width:297px;">Small gauge needles</td>
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			<td>surgery</td>
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			<td height="21" style="height:21px;">Zeiss LSM 710 NLO multiphoton microscope&#160;</td>
			<td>Carl Zeiss</td>
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			<td>Microscope</td>
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			<td height="21" style="height:21px;">Ti:Sapphire crystal laser&#160;</td>
			<td>Coherent Chameleon Ultra</td>
			<td style="width:119px;"></td>
			<td>140fs pulses of NIR light</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Zen 2010</td>
			<td>Carl Zeiss</td>
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			<td>Acquistion Software</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Imaris Bitplane&#160;</td>
			<td style="width:171px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td>Analysis Software, 3D automatic tracking</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PBS 1X</td>
			<td style="width:171px;">D. Dutscher</td>
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			<td>surgery</td>
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			<td height="21" style="height:21px;width:297px;">Thermostated chamber</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>intravital imaging</td>
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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 identification of parenchymal bone marrow niches between the bone matrix and the vessels 14. ECFP cells are distributed in the two compartments of the bone marrow, but are absent from the bone matrix. Monocytes are manually classified according to their location within the tissue. Vascular monocyte is defined when the whole cell is within the vasculature, disregarding the size of the vessel. Only big collecting venules are excluded from our analysis. Parenchymal monocyte is defined when the cell is located between the vasculature and the bone matrix.
Time-lapse imaging of specific region of interest (supplementary video 1 and 2) allows for the calculation of individual cell trajectory over time for either vascular (Figure 2A) or parenchymal (Figure 2B) monocytes. The track length shows that parenchymal monocytes display reduced displacement compared to vascular monocytes. Analysis of the displacement over time compares the mean velocity of monocytes in both compartments (Figure 2C). The mean square displacement as a function of time is a measure of the spatial extent of random motion. Motility coefficient is determined by the slope of the linear part of the curve and provides a better idea of the cell displacement (see discussion section). The motility coefficient of vascular monocytes (MC=71&#181;m&#178;/s) is higher than the motility coefficient of parenchymal monocytes (MC=4.4&#181;m&#178;/s).
The time resolution of acquisition must be adapted to the average cell velocity. Rolling vascular monocytes can make important displacement within a short time delay. For a more accurate cell tracking, reducing the time interval of acquisition is preferable. Figure 3 shows an illustration of monocyte tracking with two different time-resolutions. A 30 sec time-interval reduces the accuracy as well as the length of the pathways (in green dashed line) compared to 10 sec&#160;time-interval.
Finally, Figure 4 illustrates the possibility to analyze another cell subset concomitantly to monocytes. Ly6G is a specific marker of mature mouse neutrophils. The injection of anti-Ly6G combined with a fluorochrome (in this case Phyco-erythrin) 5 min before the imaging sequence stain neutrophils directly in vivo. This approach provides an additional dimension of analysis and the possibility to compare the behaviour of the different cell subsets. Such approach could be used to define subsets in the monocyte compartments.
<img alt="Figure 1" src="/files/ftp_upload/52476/52476fig1highres.jpg" /> 
Figure 1.&#160;Wide field of skull bone marrow organization. In vivo two-photon laser scanning microscopy image of skull steady-state bone marrow from MacBlue mouse. Vasculature (in red) is stained by tail vain injection of 2MDa Rhodamin-Dextran 5 min&#160;before imaging session. The bone is visualized by second harmonic generation (blue). ECFP+ monocytes (Cyan) are located within parenchymal niches (dark areas between vasculature and bone) and within the vessels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52476/52476fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52476/52476fig2highres.jpg" /> 
Figure 2.&#160;Real time cell tracking. Representative track paths along time of monocytes within (A) the vasculature and (B) the parenchyma. Tracks (green line) for each cell are automatically generated by the software. Red spots represent the centre of mass of tracked cells. (C) Mean velocity comparison between vascular and parenchymal monocytes. Mann-Whitney statistical analysis has been performed, *** represents p&#60;0,001. (D) Mean squared displacement (msd) as a function of time is represented for vascular and parenchymal monocytes. Asymptotic curve indicates the constrained displacement of the cells over time, whereas linear curve indicates random walk in a non-constrained environment. Motility coefficient indicates the extent of cell displacement and is determined by the slope of the curve calculated by linear regression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52476/52476fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52476/52476fig3highres.jpg" /> 
Figure 3.&#160;Time interval resolution in the study of rolling monocytes. Representative image sequences show that increased time resolution improves precision of the rolling cell trajectory. (Up) Image sequence with 10 sec&#160;time intervals. (Down) Image sequence with 30 sec&#160;time intervals. The quality of the track path is improved and the risk of considering two different cells for the same or the opposite is reduced. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52476/52476fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52476/52476fig4highres.jpg" /> 
Figure 4.&#160;In vivo co-localization of medullar neutrophils and monocytes. This figure illustrates the possibility to detect another cell subset in vivo concomitantly with monocytes, by injecting specific monoclonal antibody combined with a fluorochrome. 2 &#181;g of Ly6G-PE antibody has been injected intravenously 5 min before skull bone marrow imaging of the MacBlue mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52476/52476fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary video 1. Migratory behavior of parenchymal monocytes. In vivo 3D-imaging of the steady-state migratory activity of monocytes in the skull bone marrow parenchyma of a MacBlue mouse. The ECFP+ signal is in cyan. Migratory paths for each cell (red dot) appear in green.
Supplementary video 2. Migratory behavior of vascular monocytes. In vivo 3D-imaging of the steady-state migratory activity of monocytes in the skull bone marrow vasculature of a MacBlue mouse. The ECFP+ signal is in cyan. (2M&#160;Da) rhodamine-dextran was injected before the imaging session to distinguish the bone marrow vasculature (red). Migratory paths for each cell (red dot) appear in green.

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

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

Nanyang Technological University

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

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

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 ...

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 ...

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 ...

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 ...

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, ...

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. ...

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, ...