The authors would like to thank the Advanced Optical Microscopy Facility (www.aomf.ca) at the University Health Network for assistance with microscopy, and Mr. Jason Ellis from the Princess Margaret Cancer Center Machine Shop for manufacturing the WC and the imaging stage. We would also like to thank Dr. Iris Kulbatski for manuscript editing.

NOTE: All animal work was carried out under protocol #2615 approved by the University Health Network Institutional Animal Care and Use Committee.
1. Surgical Preparation of Mouse
<ol>
	<li>Prior to surgery, sterilize all surgical instruments and the window chamber (WC) by autoclaving. Supplement the drinking water with 50 mg/kg body weight of amoxicillin 2 days prior to surgery. Inject the mouse with 0.1 mg/kg body weight of buprenorphine intraperitoneally 4 hr prior to surgery.</li>
	<li>Anesthetize the athymic nude mouse by intraperitoneal injection of 350-400 &#956;l saline solution containing 80 mg/kg ketamine and 13 mg/kg xylazine (Note: This&#160;was approved by the Institutional Animal Care Committee, and we have not had any experience with adverse effects). Confirm proper anesthesia by testing for foot reflex. Continue observing for respiratory pattern, mucous membrane color and foot reflex during the procedure.</li>
	<li>Perform the surgical procedures under a biosafety cabinet to maintain sterile conditions during the surgery. Place the mouse on an electric heating pad covered with a surgical drape to maintain body temperature during the surgery. Perform all surgical procedures under the stereomicroscope.</li>
	<li>Apply eye ointment to keep the eyes moist during surgery. Repeat if necessary during the surgical procedure. Sterilize the operating area of the mouse by wiping with 7.5% povidone-iodine, 70% isopropyl alcohol and 10% povidone-iodine consecutively, and repeat this procedure twice.</li>
	<li>Make a 10 mm longitudinal skin incision using scalpel with a blade (#15), approaching the femur from the lateral side. Expose the femur between the flexor and extensor muscles by blunt dissection using forceps and hemostats to ensure muscle functionality.</li>
	<li>Insert the U-shaped bar under the bone, and install the WC (Figure 1). Secure the bar with the window using two nuts, and tighten the nuts with forceps. Fill the space between the bone and the WC with dental cement.</li>
	<li>Grind down the cortical bone in a 6 x 2 mm2 region with a micro-drill to gain optical access to the marrow cavity. 
	Note: Carefully inspect thinning of the cortical bone under the microscope while performing this procedure. Leave an intact, almost transparent periosteal layer of the bone.</li>
	<li>Place the 8 mm coverglass onto the window, and secure the coverglass with a snap ring to keep it in place.</li>
	<li>Readjust the musculature of the flexors and extensors back to their original locations by using forceps to maintain their function following the surgery. Suture the dermis using 5-0 suture and a needle holder.</li>
	<li>Monitor the mouse for recovery, until it is conscious and is able to move freely. Put the animal into a new cage when recovered.</li>
	<li>After recovery, perform image acquisition on the animal on the same day or the following days (see section 2). Monitor the physical condition of the mouse daily. 
	Note: Monitor physical conditions, such as weight bearing on limb, hunching, rapid or shallow breathing, hind limb swelling, self-mutilation, cellulitis and severe dermatitis.</li>
	<li>Give intraperitoneal injection of 0.1 mg/kg body weight of buprenorphine for 3 days, and 1 mg/kg body weight of meloxicam for 5 days following the surgery for treatment of post-surgical pain and inflammation. Continue providing the amoxicillin-supplemented water for 5 days.</li>
	<li>At the experimental endpoint, euthanize the animal by CO2 narcosis followed by a secondary method (e.g., cervical dislocation).</li>
</ol>
2. Image Acquisition Using Combined Multi-photon and Confocal Microscopy&#160;
<ol>
	<li>Prior to imaging, anesthetize the mouse by intraperitoneal injection of a 350-400 &#956;l saline solution containing 80 mg/kg ketamine and 13 mg/kg xylazine. Alternatively, use inhalable anesthetic, when available. Apply eye ointment.</li>
	<li>Inject the mixture of intravascular dyes for labeling vasculature (0.65 mg of 2 MDa Fluorescein (FITC)-dextran) and neutrophils (4 &#956;g of Allophycocyanine (APC)-anti-Ly6G antibody).</li>
	<li>Use an upright combined multi-photon and confocal microscope equipped with a multi-photon laser ranging from 705-980 nm for two-photon excitation, as well as single photon lasers that provides excitation wavelengths from blue to red.</li>
	<li>Set a custom-made imaging stage onto the microscope (Figure 1B). Secure the mouse on the stage by clamping the two ends of the WC with alligator clips. 
	Note: Critical components of the stage are: two alligator clips to hold the WC, and a heating element to maintain physiological temperature (37 &#176;C) of the mouse during imaging.</li>
	<li>Turn on the two-photon laser and the confocal system, and launch the image acquisition software by clicking &#34;Start System&#34;. A window with 4 tabs (Locate, Acquisition, Processing, and Maintain) will open.</li>
	<li>Set up the imaging channels. Click &#34;Smart Setup&#34; under the Acquisition tab and choose the dyes that will be used for imaging: FITC (488 nm excitation, 493-588 nm emission) and APC (633 nm excitation and 638-743 nm emission). Choose &#34;Best Signal&#34; and click &#34;Apply&#34;.</li>
	<li>Add the second harmonic generation (SHG) image acquisition channel manually. Turn on the two-photon laser under the &#34;Laser&#34; window in the Acquisition tab. Under the Channels window, click on the &#34;+&#34; sign to add a track.</li>
	<li>Set up the SHG track. Change the wavelength to 840 nm under Channels window using the up and down arrows, and choose the detection range to be 415-425 nm under &#34;Light Path&#34; window using the slider bar.</li>
	<li>Click on &#34;Oculars Online&#34; under the Locate tab. Click FITC under the filter setting for FITC to view the region of interest and adjust the focus manually by turning the focus knob on the side of the microscope. When the region of interest is located, click &#34;Oculars Offline&#34; under the Locate tab.</li>
	<li>Click on the Acquisition tab. Select FITC track under Channels window and click on &#34;Live&#34; to preview the image of FITC-dextran with 5X objective. Adjust the focus if necessary, and click &#34;Stop&#34; under Acquisition tab to stop the preview.</li>
	<li>Manually turn to 20X water objective by moving a wheel above the lenses. View the image again for FITC-dextran using the 20X objective, and adjust Master Gain and Pinhole under the Channels window to achieve optimal signal and contrast. Repeat for other channels by selecting APC and SHG tracks under the Channels window.</li>
	<li>Choose the imaging parameters. Click on &#34;Acquisition Mode&#34; to open its window. Set the frame size to be 1,024 x 1,024 pixels, and averaging of 4X to obtain high quality images.</li>
	<li>Acquire a 2D snapshot. Select all channels under Channels window and click on &#34;Snap&#34; under the Acquisition tab. Check the quality of the image and change imaging parameters if necessary.</li>
	<li>Acquire 2D time-lapse image.
	<ol>
		<li>Check off &#34;Time Series&#34; in the Acquisition tab to open the Time Series window. Type in 0 under the intervals, and 40 for total number of scans to take 40 images continuously without time interval. 
		Note: 40 scans generates approximately 5-minute long time-series with a scan speed of 7.5 sec/frame and the image size of 600 &#956;m x 600 &#956;m. Adjust the interval and number of scans depending on the microscope, image acquisition settings and the frame rate of the camera. A fixed interval time (&#62;0 sec) may be used to compare time-lapse images acquired at different time points. A longer time-lapse imaging duration may be required depending on the expected average velocity of neutrophils (or other cells of interest) in the experimental condition.</li>
	</ol>
	</li>
	<li>Acquire 3D image.
	<ol>
		<li>Check off &#34;Z-Stack&#34; in the Acquisition tab to open the Z-Stack window. Start the &#34;Live&#34; preview mode for FITC-dextran. Focus on the top of the sample and click &#34;Set First&#34;, and focus on the other end of the sample and click &#34;Set Last&#34; in the Z-stack window.</li>
		<li>Set the interval to be 10 &#956;m under the Z-stack window. Depending on the first and the last optical slice selected in the previous step, this will generate 10-20 slices, with overall optical thickness of 90-180 &#956;m. 
		Note: The z-stack interval is dependent on the size of the pinhole, which determines the thickness of the optical slice. Set the interval to be less than the thickness of the optical slice.</li>
		<li>Click &#34;Start Experiment&#34; under the Acquisition tab to start collecting images.</li>
	</ol>
	</li>
	<li>Acquire 3D time-lapse image.
	<ol>
		<li>Check off &#34;Time Series&#34; and &#34;Z-stack&#34; in the Acquisition tab to open the Time Series and the Z-stack windows.</li>
		<li>Set up the Time Series and Z-stack acquisition parameters as described in steps 2.14 and 2.15. Click &#34;Start Experiment&#34; under the Acquisition tab to start collecting images.</li>
	</ol>
	</li>
	<li>Monitor the mouse during imaging to make sure it is stable and unconscious. If the mouse is unstable and/ or conscious during imaging, remove the mouse from the stage, inject additional 100-150 &#956;l of the anesthetic solution intraperitoneally, and continue imaging. 
	Note: Signs of instability include fast breathing and motion of the mouse, which can be seen as shifting artifacts in acquired images.</li>
	<li>After image acquisition, take the mouse off the stage and return it to the cage. Use a heating lamp to keep it warm, and monitor the mouse until it is conscious.</li>
</ol>
3. Image Analysis and Quantification
<ol>
	<li>Use image analysis software to analyze the 3D and time-series data. Open the software and add images by dragging them into the open screen, which is called &#34;Arena&#34; view. Double click on an image file to open it; the image will be opened under &#34;Surpass&#34; view.</li>
	<li>Create a 2D/3D image.
	<ol>
		<li>From the &#34;More&#34; bar found on the top right corner, click &#34;Edit/ Show Display Adjustment&#34; to open &#34;Display Adjustment&#34; window. Optimize the fluorescence display by adjusting the thresholds for each channel, and take a snapshot to save the image.</li>
	</ol>
	</li>
	<li>Generate object tracks using time-lapse images.
	<ol>
		<li>Open the image to be analyzed under &#34;Surpass&#34; view. Click on the icon with an image of orange dots, found below the &#34;Surpass&#34; icon. This will open a new &#34;Spots&#34; window.</li>
		<li>Check off &#34;Track Spots (over Time)&#34; and click on the blue arrow to go to the next step. Select the channel containing the objects to track under &#34;Source Channel&#34;, and set the estimated object diameter under &#34;Estimated XY Diameter&#34; by typing a number in. Click on the blue arrow to go to the next step. 
		Note: For neutrophil tracking, select the channel containing APC fluorescence for neutrophils and set the object diameter to 12 &#956;m based on the known size of neutrophils.</li>
		<li>In the window, find and select &#34;Quality&#34; filter by clicking on the down arrow. Observe the spots defined by the software and adjust the threshold manually to include or exclude spots. Once threshold is defined, click on the blue arrow to go to the &#34;Tracking&#34; step.</li>
		<li>Under Algorithm, find and select &#34;Autoregressive Motion Algorithm&#34; by clicking on the down arrow. Type in 10 &#956;m for &#34;Max Distance&#34; and 3 for &#34;Max Gap Size&#34; found under Parameters. Check off &#34;Fill gaps with all detected objects&#34; to start tracking, and click on the blue arrow to go to the next step. 
		Note: &#34;Autoregressive motion algorithm&#34; is used for objects that have directed movement, since it generates tracks based on predicted location from previous motion. &#34;Max Distance&#34; and &#34;Max Gap Size&#34; may be adjusted depending on the expected dimension and velocity of the object being tracked, as well as the overall duration of the time-lapse image.</li>
		<li>Under Filter Type, select the &#34;Track Duration&#34; filter. Click on the green arrow to finish tracking, and check the tracks that were generated. Go back to the previous step if necessary to manually sort the tracks.</li>
	</ol>
	</li>
	<li>If the generated time-lapse image drifts, correct for translational drift.
	<ol>
		<li>Click on the icon with an image of a pencil. This is an editing tab that opens &#34;Edit Tracks&#34; window. Examine the time-lapse image and find a reference spot that should not be moving for all the images.</li>
		<li>In the left upper corner of the screen, find &#34;Pointer&#34; window and check off &#34;Select&#34; to activate the cursor for selecting a spot. Click on the reference spot.</li>
		<li>Click &#34;Correct Drift&#34; in the &#34;Edit Tracks&#34; window. Check off &#34;Translational Drift&#34;. The software will automatically generate the corrected series of images.</li>
	</ol>
	</li>
	<li>Measure cellular movement defined by the object tracks in time-lapse images.
	<ol>
		<li>Under the &#34;Edit Tracks&#34; window, check off &#34;Tracks&#34; and select a track that represents the movement of a cell. The selected track will be highlighted in yellow in the time-series image.</li>
		<li>Click on the icon with an image of a graph to open the &#34;Statistics&#34; window (statistics tab).</li>
		<li>Under &#34;Selection&#34;, select &#34;Track Speed Mean&#34; by clicking on the down arrow to generate the track speed mean for the selected track. Choose different tracks and repeat the steps to measure the track speed mean for all cells of interest.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

Real-time, serial imaging of the dynamic cellular processes in bone marrow provides information that is otherwise challenging to obtain using conventional techniques such as histology and total blood counts. The femur WC model described here provides unique opportunities to investigate cellular and structural alterations in the bone marrow over time. Although a femur WC model has been previously reported, our novel design provides a larger imaging field of view and smaller overall WC size, which are more compatible for use in adult mice. The femur WC model can be used in a variety of mouse strains including immunocompetent and/or transgenic reporter mice.
There are critical steps during the surgery that determine the overall duration and quality of imaging. When exposing the femur, only a blunt dissection of muscles between flexors and extensors is performed; removal of these muscles may result in reduced mobility of the mouse and must be avoided. Furthermore, grinding of the cortical bone must be performed carefully. Grinding approximately 50 &#956;m is adequate for optical imaging while maintaining the structural integrity of the femoral vascular network. Application of dental cement around the bone is crucial for sealing the surgically exposed area, preventing fluid accumulation, and maintaining a clear optical imaging window for over a month while reducing the risk of infection. The dental cement also acts to fix the WC in place, preventing the WC from rotating around the femoral bone. Lastly, the snap ring used to hold the coverglass is suitable for use with water-immersion lens, providing water-seal. The snap ring is recommended over adhesives to attach the coverglass to the WC because addition of adhesives may increase the gap between the bone and the coverglass, or place the coverglass at a slight angle relative to the imaging plane, thereby reducing the achievable penetration depth for imaging. Furthermore, the snap ring is useful when the coverglass needs to be cleaned or changed.
In addition to the surgery, there are several critical steps to achieve successful optical imaging of the bone marrow. To image inside the femur WC, the WC is placed horizontally to the imaging stage for proper immersion of the objective lens. The custom-made imaging stage is suitable for this purpose as it allows for adjustments of the angle of the WC with respect to the lens. Moreover, the design of the WC, particularly the size of the U-shaped bar, maintains the bone in a horizontal position as close to the coverglass as possible to achieve optimal penetration depth. During imaging, breathing artifacts may be observed as horizontal lines in the fluorescence images. In addition to securing the WC to the imaging stage, the temperature of the heating element under the imaging stage may be adjusted to adequately warm the animal to minimize excessive breathing. A thin layer of gauze may be placed between the mouse and the heated stage to further help reduce breathing artifacts during imaging. Depending on the biological imaging target, acquisition parameters such as frame rate, imaging volume and imaging duration may also be adjusted to collect sufficient images to address experimental questions. For example, velocity of neutrophil migration and crawling may differ under various experimental conditions, from 2.8 &#956;m/min for resident neutrophils under basal conditions to anywhere ranging from 5-10 &#956;m/min for neutrophils under inflammatory conditions19-21. Thus, the appropriate imaging speed and duration for neutrophil tracking in vivo would also differ based on experimental conditions. In addition, as seen from the results, tracking of cells within the vasculature would require a fast imaging camera with a high frame rate (30 frames/sec). However, if the target cells are residing in extravascular space, or if the cell-to-cell interaction of interest is expected to occur more slowly, image quality should be prioritized over imaging speed.
The influence of injected antibodies for in vivo cell staining on cellular function must also be considered. In our study, we used anti-Ly6G antibody (1A8 clone), which is typically used at high doses (150-250 &#956;g) to deplete neutrophils from circulation and study their role in various disease models. Yipp and Kubes demonstrated that fluorochrome-labeled anti-Ly6G antibody does not disrupt leukocyte recruitment at low intravascular injection doses (1-40 &#956;g)22. However, a more recent study suggests that fluorochromes, having great differences in molecular mass and chemical structure, can differentially influence the function of anti-Ly6G 23. The study demonstrated that FITC-labeled anti-Ly6G, but not PE- and APC-labeled antibodies, effectively depletes neutrophils in vivo at a low concentration (30 &#956;g). In our study, we did not observe neutrophil depletion overtime using 4 &#956;g of APC-labeled anti-Ly6G. Nevertheless, further characterization of the function of fluorochrome-labeled anti-Ly6G antibody in vivo is required to validate its use in intravital imaging. This also applies to other antibodies that have functional ability to alter cellular interactions. As an alternative to in vivo cell staining with antibodies, transgenic reporter mice24 and/or labeled fluorescent cells25 can be used to visualize neutrophils or other cell population of interest in vivo.
The femur WC model described in this protocol enables imaging for up to 40 days after the surgery. Some of the challenges for long-term imaging using this WC model include the delayed bone healing process and maintenance of a clean glass window. Notably, we did not observe signs of obvious bone healing following the WC implantation, which would include thickening of periosteum; this is not unexpected given that bone healing process typically occurs after 1 &#189;-3 months26,27. Maintenance of a clean and optically-clear window can be achieved by the application of the dental cement, and the use of snap ring and coverglass which provide a firm water-seal with the tissue. The coverglass can be occasionally cleaned with cotton tips from the outside to remove dust and debris. The WC has a wide imaging field, almost covering the entire length of the diaphysis region (approximately 8.4-8.6 mm28), which makes it superior to the previously published model18. However, access to the distal regions and the deeper marrow cavity is limited. In particular, the trabecular-rich metaphysis, located at the distal ends of the diaphysis, is known to be rich in HPSCs29. These areas may be accessed histologically, or by IVM as a terminal procedure. Alternatively, different imaging modalities such as micro-computed tomography can be used to image the distal regions of the bone marrow in vivo30. Such methods are complementary to IVM, providing an integrated approach for assessing anatomy and the various cellular and structural alterations and interactions within the bone marrow.
The protocol described here offers a novel approach for long-term spatio-temporal assessment of the femoral bone marrow in mice at cellular resolution. The methods presented enable in vivo serial imaging to track movements of a single cell or cellular populations over biologically relevant time scales, while simultaneously obtaining structural and functional information of the bone marrow. In addition, quantitative image analysis can be performed to further characterize cellular and functional alterations, such as tracking of cellular movements. Application of this femur WC model in various strains of mice, particularly in transgenic reporter mice31, may further enable investigation of multiple individually-labeled cell populations such as those involved in hematopoiesis. Therefore, the described protocol is widely applicable to a wide range of preclinical studies involving various biological processes within the living murine bone marrow, including investigations in hematopoiesis, leukemia, HPSC transplantation, immune regulation, the bone marrow microenvironment (e.g., hypoxic niche) and the contribution of immune cells to tumor progression and metastasis.

Bone marrow is an important organ involved in hematopoiesis and immune regulation. It consists of a hematopoietic component containing hematopoietic stem and progenitor cells (HSPCs), and a stromal component containing non-hematopoietic progenitor cells that give rise to mesenchymal cells1. Two-thirds of hematopoietic activity is dedicated to the generation of myeloid cells2. In particular, a large number of neutrophils are produced in the bone marrow, with 1-2 x 1011 cells generated per day in a normal adult human2. Neutrophils are the first line of defense against microbial infections and are mostly reserved in the bone marrow until stress triggers their mobilization to supplement peripheral neutrophils1,3. In addition to their anti-microbial effects, recent studies suggest an important role of neutrophils in cancer biology, having both pro- and anti-tumorigenic phenotypes depending on transforming growth factor beta (TGF-&#946;) signaling in the tumor microenvironment4,5. Moreover, studies demonstrated that neutrophils that accumulate in primary tumors exert pro-tumorigenic and metastatic effects by suppressing the cytotoxic function of T cells6,7, while neutrophils in circulation exert a cytotoxic, anti-metastatic effect8. As such, investigation of hematopoietic cells in the bone marrow, particularly neutrophils, is crucial to elucidating their role in immune and tumor regulation.
Histopathology and a complete peripheral blood count are routinely used to evaluate cellular and structural alterations in the bone marrow9. However, these methods only provide static information of different cell populations or tissue microstructures. Longitudinal in vivo imaging can be used in combination with the standard methods to assess the dynamics of multiple cellular, vascular and stromal components as well as cell-to-cell interactions in a longitudinal manner. Intravital microscopy (IVM), defined as imaging of living animals at microscopic resolution10, is particularly useful for assessing dynamic cellular processes over time in the same sample, reducing the number of experimental animal required. IVM is often combined with a chronically transplanted window chamber (WC) to access the organ of interest for imaging over a duration of weeks to months. Cranial and dorsal-skinfold WC models have the longest history of use dating back to the mid 1990s. More recently, other organ-specific WC models such as those of the mammary fat pad and various abdominal organs have been developed11.
The typical approach for imaging bone marrow in vivo has mainly involved exposure of the calvaria of mice, where the thinned bone enables direct visualization of single cells with minimal surgical intervention12-14. However, the calvarial bone marrow may be distinct from that of other bones, such as the long bone, as demonstrated by a lower number of HSPCs and hypoxic cells in calvaria, which indicates reduced maintenance and development of HSPCs15. Therefore, alternative approaches for assessing cellular components in the long bone have been investigated. These include direct exposure of the femoral bone marrow16 and ectopic transplantation of split femur in the dorsal skinfold WC17. However, the former is a terminal procedure that does not permit tracking of cellular, structural and functional alterations over longer time periods, and the latter likely disturbs normal bone marrow function due to the transplantation of femur to an ectopic site inside the dorsal skinfold WC. Another method that enables orthotopic serial imaging of femoral bone marrow over time is the use of a WC in the femoral bone. One previous report demonstrated long-term imaging of microcirculation in the femoral bone marrow using a femur WC in mice18. Additionally, the authors demonstrated visualization of tumor cells in the femur, indicating its utility in monitoring bone marrow metastasis. However, this WC design was limited by its large size (1.2 cm diameter) and relatively small imaging area (4 mm diameter), which was only suitable for large mice (26-34 g, 3-6 months of age) thereby making the approach impractical for routine use.
Therefore, a new WC with a smaller overall size and larger inner imaging area was designed for the purpose of this study. The goal of this study was to provide a method for imaging various cell types in the femoral bone marrow. The femur WC model was developed in-house and was used to visualize and track neutrophils within the 3D vascular network. Using this model, IVM of the bone marrow can be performed serially over 40 days. This approach can be applied to a variety of fields for elucidating the processes of hematopoiesis, immune regulation and tumor development.

<table><tbody><tr><td>NRCNU-F athymic nude mice</td><td>Taconic</td><td>Ncr nude</td><td>8-10 weeks old, female</td></tr><tr><td>Saline</td><td>Baxter</td><td>JB1302P</td><td></td></tr><tr><td>Ketamine hydrochloride</td><td>Bioniche Animal Health Canada, Inc.&amp;nbsp;</td><td>DIN 01989529</td><td></td></tr><tr><td>Xylazine</td><td>Bayer HealthCare, Bayer Inc.</td><td>DIN 02169592</td><td></td></tr><tr><td>Surgical drape</td><td>Proxima</td><td>DYNJP2405</td><td></td></tr><tr><td>Electric heating pad</td><td>Life Brand</td><td>57800827375</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>Leica M60</td><td></td></tr><tr><td>Eye ointment (tear gel)</td><td>Novartis&amp;nbsp;</td><td>T296/2</td><td></td></tr><tr><td>7.5% betadine</td><td>Purdue Frederick Co</td><td>67618-151-16</td><td></td></tr><tr><td>70% isopropyl alcohol</td><td>GreenField</td><td>P010IP7P</td><td></td></tr><tr><td>10% betadine</td><td>Purdue Frederick Co</td><td>67618-150-05</td><td></td></tr><tr><td>Scalpel handle (#3)</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Scalpel blade (#15)</td><td>VWR</td><td>89176-368</td><td></td></tr><tr><td>Spring Scissors curved</td><td>Fine science Tools</td><td>15023-10</td><td></td></tr><tr><td>Baby-Mixter Hemostat</td><td>Fine science Tools</td><td>13013-14</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine science Tools</td><td>14094-11</td><td></td></tr><tr><td>Extra Fine Graefe Forceps</td><td>Fine science Tools</td><td>11151-10</td><td></td></tr><tr><td>Halsted-Mosquito Hemostats</td><td>Fine science Tools</td><td>13008-12</td><td></td></tr><tr><td>Micro-drill</td><td>Harvard Apparaus</td><td>72-6065</td><td></td></tr><tr><td>Micro-drill burrs</td><td>Fine Science Tools</td><td>19007-14</td><td></td></tr><tr><td>Femur window chamber</td><td>PMCC machine shop</td><td>custom design</td><td>9.1 mm- 8.5 mm- 7.5 mm (outer to inner diameter), 2.16 mm (radius of two holes), 13.9 mm (distance between two holes), 0.7 mm (thickness)</td></tr><tr><td>U-shaped bar</td><td>PMCC machine shop</td><td>custom design</td><td>13.8 mm (length), 1.6 mm (width), 3.7 mm (height)</td></tr><tr><td>Coverglass (8 mm)</td><td>Warner Instruments&amp;nbsp;</td><td>HBIO 64-0701 CS-8R</td><td></td></tr><tr><td>Retaining ring (8 mm)</td><td>ACKLANDS GRAINGER</td><td>UNSPSC # 31163202</td><td></td></tr><tr><td>Nuts (hexagon stainless steel)</td><td>Fastenal</td><td>70701</td><td></td></tr><tr><td>Dental cement</td><td>3M</td><td>RelyX U200</td><td></td></tr><tr><td>Suture (5-0 Monosof black)</td><td>Covioien</td><td>SN-5698</td><td></td></tr><tr><td>Halsey needle holder</td><td>Fine Science Tools</td><td>12501-13</td><td></td></tr><tr><td>Buprenorphine (Temgesic)</td><td>Reckitt Benckiser</td><td>DIN 0281251</td><td></td></tr><tr><td>Meloxicam (Metacam)</td><td>Boehringer Ingelheim</td><td>DIN 02240463</td><td></td></tr><tr><td>Amoxicillin (Clamavox)</td><td>Pfizer</td><td>DIN 02027879</td><td></td></tr><tr><td>FITC-Dextran</td><td>Sigma-Aldrich</td><td>FD2000S</td><td></td></tr><tr><td>APC- Anti-Mouse Ly-6G (Gr-1)&amp;nbsp;</td><td>eBioscience</td><td>17-9668</td><td></td></tr><tr><td>Two-photon microscope LSM 710</td><td>Carl Zeiss</td><td>Zeiss LSM 710 NLO</td><td></td></tr><tr><td>Imaging stage</td><td>PMCC machine shop</td><td>custom design</td><td>15.9 cm (length), 11 cm (width), 0.9 cm (height)</td></tr><tr><td>Imaris software</td><td>Bitplane</td><td>Imaris 8.0</td><td>Image analysis software described in Section 3 of the Protocol&amp;nbsp;</td></tr><tr><td>Zen 2012</td><td>Zeiss</td><td>Zen 2012</td><td>Image acqusition software described in Section 2 of the Protocol&amp;nbsp;</td></tr></tbody></table>

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.

Murine femoral bone marrow is successfully accessed using the WC to enable visualization of individual neutrophils and vascular networks. Figure 1 shows the WC instrument and describes the surgical procedure, which involves exposure of the femoral bone and thinning of cortical bone to gain optical access inside the bone. The surgery is well-tolerated in mice; they were assessed for adverse physical reactions, such as hind limb swelling, non-weight bearing on limb, self-mutilation, cellulitis and dermatitis, as well as damage to the WC for 5 days post-surgery, with no report of distress. Notably, the window remains optically clear for IVM for up to 40 days, which may enable long-term assessment of cellular and structural alterations following an intervention.
Vasculature within the bone marrow can be visualized by intravascular dyes, such as FITC-Dextran (Figure 2). Since the contrast is dependent on the injection of the dye, the acquired image represents functional vasculature where there is sufficient blood flow. In addition to the vasculature, neutrophils can be stained in vivo using anti-Ly6G antibody. Figure 3A and 3B represent the same area of the bone marrow observed at different time points, where neutrophils are seen both in the intravascular and extravascular space. These areas were characterized by the vascular landmarks. Rhodamine 6G may be used as an alternative to anti-Ly6G to stain neutrophils in vivo; however, it diffuses out of the vasculature and stains monocytes in the extravascular space in a non-specific manner. Therefore, anti-Ly6G is preferred because it enables specific imaging of neutrophils. Figure 3C represents imaging of the cortical bone, which is mainly composed of collagen fibers. The maximal depth for imaging achieved in this experiment is 180 &#956;m (Figure 3C).
Time-lapse fluorescence imaging facilitates tracking and quantification of cellular movement in vivo. Figure 4A shows tracking of neutrophils in the bone marrow vascular niche. Imaging with no additional time interval between each scan allows for accurate tracking of cells; however, some user input is required to further improve the accuracy of tracks generated by the software. Tracking of spots (i.e., cells of interest) enables quantification of various parameters, such as mean track speed (Figure 4B). Other parameters that can be measured include, but are not limited to, track length, change in spot size, shape and intensity. This assay provides quantification of various parameters that improve the characterization of cellular movements and interactions.
<img alt="Figure 1" src="/files/ftp_upload/54205/54205fig1.jpg" /> 
Figure 1: Femur window chamber (WC), imaging stage and the surgical procedure. (A) Custom-made titanium femur WC with a U-shaped bar that serves to fix the femur in place. The WC holds an 8 mm diameter cover glass, secured by a snap ring. (B) Custom-made imaging stage. A heating element is placed on the bottom of the stage to maintain the physiological temperature of the mouse during imaging. The two alligator clips are used to hold the WC. (C-H) Surgical procedure for installing the femur WC. (C) The femur is exposed between the flexor and extensor muscles. Muscles are not removed during the procedure. (D) The U-shaped bar is placed under the femur, and (E) the WC is installed and secured using two nuts. (F) Dental cement is applied between the bone and the WC to fill the space. (G) The dermis is sutured and (H) the cover glass is fixed in place with the retaining ring. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54205/54205fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54205/54205fig2.jpg" /> 
Figure 2: 3D fluorescence imaging of vascular network in the bone marrow in vivo. Representative image of vasculature in the bone marrow, imaged through the femur WC. Vasculature (green) is visualized by tail vein injection of 2 MDa FITC-Dextran. Scale bar = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54205/54205fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54205/54205fig3.jpg" /> 
Figure 3: 3D imaging of vascular network, neutrophils and bone matrix in vivo. Images of vasculature and neutrophils in the bone marrow (A) 3 days and (B) 10 days after the femur WC surgery. Neutrophils, stained by tail vein injection of APC-labeled Ly6G antibody, are seen both in the intravascular and extravascular spaces. The arrows point to the vascular structure seen on both days, indicating the ability to track the same location over time. Scale bar = 50 &#956;m. (C) Bone matrix can be visualized using SHG (white), in addition to vasculature (green) and neutrophils (red). The image was acquired 40 days after the WC surgery. Scale bar = 70 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54205/54205fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54205/54205fig4.jpg" /> 
Figure 4: Tracking neutrophil movement within the vascular network of the bone marrow in vivo. (A) Track paths of neutrophils within the vascular network. Tracks are generated automatically using the software&#39;s built-in Autoregressive Motion algorithm. Each white dot represents a center point of a particle being tracked (neutrophils). Scale bar = 50 &#956;m. (B) Mean track speed measured for intravascular and extravascular neutrophils, indicating difference in the movement of neutrophils (Error bar = mean + SD, * p = 0.0176, two-tailed t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54205/54205fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow at JoVE.com

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.

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, ...

femur-window-chamber-model-for-vivo-cell-tracking-murine-bone

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Princess Margaret Cancer Centre. 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.

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

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Tracking Mouse Bone Marrow Monocytes In Vivo

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

Tracking Mouse Bone Marrow Monocytes In Vivo

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

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

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

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, ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Biology

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) ...

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. ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

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

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