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 u...

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.

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			<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.&#160;</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>
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		<tr>
			<td>Eye ointment (tear gel)</td>
			<td>Novartis&#160;</td>
			<td>T296/2</td>
			<td></td>
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		<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>9.1mm- 8.5mm- 7.5 mm (outer to inner diameter), 2.16 mm (radius of two holes), 13.9mm (distance between two holes), 0.7mm (thickness)</td>
		</tr>
		<tr>
			<td>U-shaped bar</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>13.8mm (length), 1.6 mm (width), 3.7mm (height)</td>
		</tr>
		<tr>
			<td>Coverglass (8mm)</td>
			<td>Warner Instruments&#160;</td>
			<td>HBIO 64-0701 CS-8R</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring (8mm)</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)&#160;</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>15.9cm (length), 11cm (width), 0,9cm (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&#160;</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&#160;</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-mu...

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

Research

JoVE Journal

Immunology and Infection

8.1K 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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

Tracking Mouse Bone Marrow Monocytes In Vivo

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

Tracking Mouse Bone Marrow Monocytes In Vivo

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

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

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

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

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

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

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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

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

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

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

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...