We would like to thank Ying Yu for ApcMin/+ mice genotyping. This study was supported by funds from INSERM and grants (CA057621 and AI053194) from the National Institutes of Health.

NOTE: All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC), UCSF. All the imaging experiments were non-survival procedures and the animals were euthanized immediately following the end of image acquisition.
1. Generation of Mice
NOTE: ApcMin/+ mice, carrying a mutation on the Apc gene, spontaneously develop 50-100 adenomas in the small intestine.
<ol>
	<li>Cross ApcMin/+ mice with the ACTB-ECFP line, which expresses ECFP under the actin promoter, and further with the c-fms-EGFP line, which expresses EGFP under the c-fms promoter to detect the myeloid cells. Heterozygous animals for ACTB-ECFP and c-fms-EGFP are used to detect fluorescence.</li>
	<li>Use mice at 3-4 months of age to image clearly detectable adenomas.</li>
</ol>
2. Preparation of Materials Before Surgery
NOTE: The details of the microscope set-up have been previously described6,7.
<ol>
	<li>Microscope
	<ol>
		<li>Turn on the heating blanket. Turn on the argon laser for 488 nm excitation and the solid-state 405 nm, 561 nm, and 640 nm lasers. Turn on the microscope, the spinning disk control unit, the AOTF, the laser control unit, and the camera controller.</li>
		<li>Open the microscope shutter, which turns the LED red. Turn on the computer and camera software.</li>
	</ol>
	</li>
	<li>Imaging Platform
	<ol>
		<li>Ensure that the stage insert is clean. Otherwise, clean with soap and water and then dry.</li>
		<li>Take two coverslips and use lab tape to secure them on the insert. Place the insert on the stage and clean it with alcohol wipes.</li>
		<li>Using lab tape, attach the heating blanket to the right side of the stage.</li>
		<li>Verify the integrity of the rubber diaphragm on the nose cone and change it if necessary. Position and fix the nose cone for isoflurane anesthesia delivery to the animal by using gauze and lab tape.</li>
	</ol>
	</li>
	<li>Isoflurane Anesthesia System
	<ol>
		<li>Refill the isoflurane tank.</li>
		<li>Turn on the vacuum and adjust to 1.2 L/min.</li>
		<li>Add distilled water to the nebulizer and connect the nebulizer to the isoflurane system.</li>
		<li>Turn on the oxygen analyzer and connect it to the isoflurane system. Turn on the oxygen tank and ensure that the oxygen analyzer shows 100%. Adjust the O2 flow to 0.2 L/min.</li>
		<li>Turn on the nitrogen tank and adjust the flow at 0.8 L/min. Ensure that the oxygen analyzer shows 21%.</li>
	</ol>
	</li>
	<li>Preparation of Surgery Tools and Platform
	<ol>
		<li>Clean 2 pairs of dissection forceps and scissors with soap and water.</li>
		<li>Turn on the hot bead sterilizer and let it reach 250 &#176;C. Sterilize the surgery tools for 30 sec. Let them cool off.</li>
		<li>Place a lab soaker on the surgery bench.</li>
		<li>Use a Styrofoam lid as a surgical platform. Cover with a piece of lab soaker. Prepare a second piece of lab soaker to change after shaving the mouse.</li>
		<li>Place the nose cone with the line of anesthesia on the covered Styrofoam lid, and attach it to the Styrofoam lid (not on the lab soaker) with some tape and use two needles on both sides of the nose cone to keep it in place.</li>
		<li>Assemble betadine, alcohol wipes, sterile gauze, super glue, microscope slides, electric shaver, and 4 pieces of lab tape.</li>
	</ol>
	</li>
</ol>
3. Preparation of Injections
<ol>
	<li>Under the hood, prepare an insulin syringe (28 G x &#189; in) with 7 &#181;l of stock AF647-conjugated Ly-6G (Gr1) antibody (1 mg/ml) into 100 &#181;l of 2,000 kDa rhodamine-dextran (4 mg/ml) for retro-orbital injection. Retro-orbital injection is recommended in ApcMin/+ mice since anemia that develops in these animals makes the tail veins difficult to detect through the skin.</li>
	<li>Prepare a second insulin syringe with 1 mg/kg atropine diluted into 150 &#181;l PBS for subcutaneous (s.c.) injection before imaging to dampen peristaltic movements of the intestine.</li>
</ol>
4. Preparation of the Intestine for Imaging
<ol>
	<li>Verify that the anesthesia line to the induction chamber is open, and the lines to the surgical table and microscope are closed.</li>
	<li>Transfer the animal to the anesthesia chamber. Close the anesthesia chamber and turn on the isoflurane to 4% (with 21% O2).</li>
	<li>When the mouse breathes deeply and slowly (after 5-6 min), transfer it to the surgical stage on its flank and inject the Gr1 antibody solution and/or dextran solution retro-orbitally.</li>
	<li>Open the anesthesia line linked to the surgical platform. Close the line to the anesthesia chamber and put the mouse on its back with its nose in the anesthesia cone.</li>
	<li>Reduce the isoflurane concentration from 4% to 2.5%.</li>
	<li>Verify that the mouse is well anesthetized by pinching its footpad and testing the corneal reflex.</li>
	<li>Add ophthalmic lubricating&#160;ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Secure the limbs of the mouse to the surgical stage by adding lab tape.</li>
	<li>Remove the hair from the ventral surface by using an electric shaver. Also remove the hair from the left hind limb to position the oximeter before acquisition. Remove the lab soaker from the surgical stage and replace with a clean one to avoid hair contamination.</li>
	<li>Disinfect the ventral surface with alcohol wipes and betadine.</li>
	<li>Inject the atropine solution (from 3.2) s.c. in one flank of the mouse.</li>
	<li>Make a 1 cm ventral midline incision through the skin and the peritoneum by using scissors and one pair of forceps. Carefully pull out 3-4 cm of intestine and identify one or several tumors to image.</li>
	<li>Take a glass microscope slide and position a loop of the intestine on it with a tumor at the top.</li>
	<li>Add some super glue to a few locations along the intestine to attach it to the slide. Wait one min for the glue to dry. Use a marker to draw a line on the slide pointing to the tumor in order to facilitate its localization with the confocal.</li>
</ol>
5. Positioning the Mouse on the Stage and Preparation for Image Acquisition
<ol>
	<li>Remove lab tape from the limbs.</li>
	<li>Open the anesthesia line to the microscope stage and close the one to the surgical platform.</li>
	<li>Transfer the mouse carefully to the microscope stage with its ventral side down and its nose in the nose cone. Secure the anesthesia line with some lab tape.</li>
	<li>Re-position the mouse and/or the slide in order to have the intestine in the center of one of the cover-slipped windows. Gently tape down the slide with lab tape.</li>
	<li>Attach the oximeter probe to the left limb of the mouse to follow its heart and respiratory rate and arterial oxygen saturation levels.</li>
	<li>Cover the mouse with the heating blanket to avoid hypothermia.</li>
	<li>Decrease the isoflurane concentration from 2.5% to 1-1.5% for imaging.</li>
</ol>
6. Acquisition of Images by Using &#181;Manager Software
<ol>
	<li>Increase CSUX speed to 2,500 rpm to improve the image quality in the Configuration Settings.</li>
	<li>Use the Objective dropdown menu to choose 10X 0.5 NA or 20X 0.75 NA Fluar lens objective.</li>
	<li>Choose the laser 405 nm from Color dropdown menu.</li>
	<li>Click on Live and adjust the focus with the microscope.</li>
	<li>Click on Auto to automatically adjust maximum and minimum pixel intensities of the display image based on the extreme pixel values in the image. A histogram of display image&#39;s pixel intensities is presented in the graph in the lower part of the Main Window.</li>
	<li>Adjust the camera exposure in the Piper Control Panel window by changing the number of clocks (One clock is 33.333 msec).</li>
	<li>If the signal is too bright with the lowest exposure, decrease laser intensity using the microscope control unit.</li>
	<li>Check the signal intensity for 488 nm, 561 nm and 640 nm laser lines. Adjust laser intensity with the laser&#8217;s own control unit (488 nm and 561 nm) or the microscope control unit.</li>
	<li>In the Multi D Acquisition window, check the Time points box and type in number of time points and desired time interval.</li>
	<li>Check the Z-stacks box, choose &#8220;relative Z&#8221; and define Z-start and Z-end relative to the current position. 
	NOTE: For 10X or 20X objective, start with 3 Z planes, 8 &#181;m or 4 &#181;m apart respectively.</li>
	<li>Check the Multiple positions (XY) box for imaging several locations, then click Edit position list, mark 4 to 6 different positions.</li>
	<li>Check the Channels box and add channels from New and select lasers lines from the dropdown menu and type in exposure for each channel (in multiples of 33.333 msec).</li>
	<li>Check Save images in the &#8220;Multi D acquisition&#8221; window.</li>
	<li>Select a folder and all the images acquired will be automatically saved in this folder with the name prefix given. 
	NOTE: Each continuous acquisition will be saved in a separate subfolder as individual tiff files for each Z slice in each color at each time point.</li>
	<li>Save all the settings by clicking Save settings in the Multi D Acquisition window.</li>
	<li>Click Acquire in the Multi-dimensional Acquisition window to start acquisition.</li>
	<li>If the oximeter probe is not used or stops working well (when pulse is not stable or hard to detect), check the efficiency of anesthesia every 15 min during the imaging procedure by pinching the footpad and checking the corneal reflex. Adjust the anesthesia if the mouse is responsive to these stimuli.</li>
	<li>After acquisition, euthanize the mouse by increasing the isoflurane concentration to 5%. When no breathing movements are detectable, remove the mouse from the stage and perform a cervical dislocation.</li>
</ol>
7. Analysis of Images by Using Imaris Software
<ol>
	<li>Conversion to .ims Files (Supplementary Figure 1A)
	<ol>
		<li>Open the camera software, click on File then Batch Converter.</li>
		<li>Click on Add files and choose the first file of the first position. Repeat for each position.</li>
		<li>For each file uploaded, click on Settings and verify that the settings &#8220;t&#8221; for Time, &#8220;c&#8221; for Channels and &#8220;z&#8221; for Z-stacks appear in that order.</li>
		<li>Save in the desired folder. Browse and create a new file as converted.</li>
		<li>Click on Set for all and Start.</li>
	</ol>
	</li>
	<li>Adjustments (Supplementary Figure 1B)
	<ol>
		<li>Go into the specific Converted folder and open the first file.</li>
		<li>In Display Adjustment window, adjust the background signal cutoff by modifying the Min number for each channel&#160;if necessary.</li>
		<li>If necessary, adjust the signal intensity by modifying the Max number (the lower the Max number, the higher the intensity of the signal). 
		NOTE: Adjustments of the signal intensity/contrast enable the highlight of a cell behavior or a compartment in order to have a nicer visualization. It does not change the result itself.</li>
		<li>Click on Edit and Image Properties.</li>
		<li>Change the x, y, and z values (for a 10X objective, x and y = 0.712, z = 8 &#181;m; for a 20X objective&#160;: x and y = 0.36, z = 4 &#181;m).</li>
		<li>Click on All Equidistant to adjust time points. Add the start date and start time of the acquisition. Add the end date and end time of the acquisition (Supplementary Figure 1C).</li>
		<li>Click on Play button to watch the movie.</li>
		<li>Click Edit and Delete time points to delete blurry images.</li>
		<li>To join two separate acquisitions together into one movie, go to the last time point of the first movie and click on Edit&#160;&#62;&#160;Add time points to add the second file.</li>
	</ol>
	</li>
	<li>Saving as a movie (Supplementary Figure 1D)
	<ol>
		<li>Click on Record, choose the .mov extension and a compression factor of 0.</li>
		<li>Click on Save.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this paper, a detailed protocol is described for spinning disk confocal imaging of myeloid cell dynamics in intestinal tumors for several hours in a live animal, imaged from the serosal side of the gut.
To avoid inflammation and to have optimal physiologic conditions, imaging of the intestine has to be done on the intact organ. However, imaging from the serosal side of the intestine is challenging, since the light has to go through different tissue layers such as smooth muscle before reachi...

Overwhelming evidence now demonstrates that the tumor microenvironment, consisting of heterogeneous cell populations, including fibroblasts, endothelial cells, immune and inflammatory cells, extracellular matrix, and soluble factors, plays a crucial role in the initiation and progression of solid tumors by contributing to almost all hallmarks of cancer1. Indeed, during tumor progression, there are constant dynamic interactions between transformed cancer cells and stromal cells that evolve to generate a microenvironment favorable to malignancy2. Among the immune cells that infiltrate the tumor microenvironment, myeloid cells are the most abundant3. Consisting of tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSCs), dendritic cells (DC) and neutrophils (PMNs), myeloid cells are recruited from bone marrow and progressively infiltrate tumors, releasing cytokines, growth factors and proteases which can promote tumor growth and spread4. The crosstalk between cancer cells and myeloid cells is complex but dynamic. Thus the understanding of the nature of their interactions is crucial for determining why these cells promote cancer progression instead of participating in an anti-tumor immune response, and may help to find new targets to control it.
Direct observation by intravital microscopy provides information on cell dynamics within the tissues of live mice5. A four-color, multi-region, micro-lensed spinning disk confocal system was designed to study stromal cells within mammary tumors6. This approach enables long-term continuous imaging and includes several advantages such as (a) rapid images acquisition to minimize motion artifacts, (b) long-term anesthesia, (c) four color acquisition to follow different cell types, (d) fluorescent labeling of different tumoral components, and (e) observation of different tumor microenvironments within the same mouse to avoid mouse to mouse variability7-9. With this technology, different cell behaviors have been reported in the mammary tumor virus (MMTV) promoter-driven polyoma middle T oncogene (PyMT) model that displays progressive stages of tumorigenesis. Regulatory T-lymphocytes (Tregs, visualized by the Foxp3EGFP transgene) migrate preferentially in proximity to blood vessels whereas DCs (CD11c-DTR-EGFP), carcinoma-associated fibroblasts (Fsp1+/+-EGFP) and myeloid cells (c-fms-EGFP) exhibit higher motility at the tumor periphery than within the tumor mass. In the condition of acute systemic hypoxia, cells migrated differently: Tregs stop migrating in contrast to myeloid cells that continue to move6. In addition, in the same mouse model, it has been shown that doxorubicin sensitivity changes with tumor stage, drug distribution is related to drug response, and doxorubicin treatment leads to CCR2-dependent recruitment of myeloid cells to tumors. Thus, live imaging can also be used to gain insights into drug responses in&#160;situ and the biology of chemoresistance10,11.
Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas12 and mutation of a single copy of the Apc gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer13. The mouse strain ApcMin/+ carries a truncation mutation at codon 850 of the Apc gene and spontaneously develops multiple intestinal adenomas all over the small intestine14-16. Long-term intravital imaging of the intestine is challenging because of the invasiveness of the procedure, since opening the peritoneal cavity is necessary for access to the intestine. Short-term live imaging studies have been previously published on healthy intestine17,18, but long-term direct observation of intestinal tumors has not been reported. A surgical procedure has been designed and refined to visualize tumors through the serosal surface of the intestine, using the intravital spinning disk microscopy system previously used to image breast tumors6,10. In this paper, a protocol is described that allows one to follow the behavior of myeloid cells within the tumors in the small intestine by using ApcMin/+ mice.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;"><a name="RANGE!A1:D30">Name of Material/ Equipment</a></td>
			<td style="width:107px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:227px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ApcMin/+ mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td>2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ACTB-ECFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td style="width:141px;">3773</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cfms-EGFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td align="right">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,000 kDa Dextran, rhodamine-conjugated</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:141px;">D7139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isoflurane</td>
			<td style="width:107px;">Butler Animal Health Supply</td>
			<td style="width:141px;">29450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitrogen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1X PBS</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Saline Buffer</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse Ly-6G (GR1) antibody AF647</td>
			<td style="width:107px;">UCSF Monoclonal antibody core</td>
			<td style="width:141px;"></td>
			<td>Stock 1mg/ml. Use at 7ug/mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atropine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td>Use at 1mg/Kg mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alcohol wipes</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">326895</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">28G1/2 insulin syringe</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">329465</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Remium cover glass</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-548-5M</td>
			<td>24x50-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost plus microscope slides</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-550-15</td>
			<td>25x75x1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Krazy glue</td>
			<td style="width:107px;">Office Max</td>
			<td align="right">7111555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Betadine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heat blanket</td>
			<td style="width:107px;">Gaymar Industries</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hot bead sterilizer</td>
			<td style="width:107px;">Fine Science Tools</td>
			<td style="width:141px;">18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cotton tipped apllicators 6-inch</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:141px;">72310-10</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anesthesia system</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:107px;">Carl Zeiss Inc</td>
			<td style="width:141px;">Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">stage insert</td>
			<td style="width:107px;">Applied Sientific Instrumentation</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse Ox oximeter, software and sensors</td>
			<td style="width:107px;">Starr Life Sciences</td>
			<td style="width:141px;">MouseOx</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nebulizer</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaris</td>
			<td style="width:107px;">Bitplane</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mManager</td>
			<td style="width:107px;">Vale lab, UCSF</td>
			<td style="width:141px;"></td>
			<td>Open-source software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ICCD camera</td>
			<td style="width:107px;">Stanford Photonics</td>
			<td style="width:141px;">XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinning disk confocal sacan-head</td>
			<td style="width:107px;">Yokogawa Corporation</td>
			<td style="width:141px;">CSU-10b</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

By using spinning disk confocal microscopy, non-tumoral and tumoral tissues in the small intestine of ApcMin/+;ACTB-ECFP;c-fms-ECFP mice can be visualized from the serosal surface. After imaging, camera software is used to analyze and adjust the acquisition (Supplementary Figure 1). After intravenous (i.v.) injection of fluorescent 2,000 kDa dextran-rhodamine and a Ly-6G 647 conjugated antibody, blood vessels and PMNs can be detected respect...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

real-time-imaging-myeloid-cells-dynamics-apcmin-intestinal-tumors

Research

JoVE Journal

Immunology and Infection

9.6K Views.  INSERM U661, Functional Genomic Institute. By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. 

Video: Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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

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

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

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

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.