Nitya Jain was supported by an NIH/NIAID Career Transition Award 1K22AI116661-01.

All animal procedures were performed with the approval of and in compliance with the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital.
CAUTION: This protocol involves the use of a class 3b laser (LG3). LG3 laser safety goggles must always be used when operating this laser. Appropriate training and safety guidelines must be followed to avoid the risk of injury.
1. Design and Assembly of Laser
<ol>
	<li>Connect the light-emitting diode (LED) (405 nm, 19.3 mW, 1400 mA, fiber-coupled) to the LED driver via the 4-pin M8 connector cable that comes attached to the LED. Carefully tighten the screw to fasten the connection.</li>
	<li>Before attaching the optogenetics patch cable to the LED, hold the connector opening of the LED up to the light to ensure there are no visible obstructions. Blow on the hole to eliminate any obstructions if necessary and insert the optogenetics patch cable into the opening.</li>
	<li>Connect the fiber optic cannula to the optogenetics patch cable by inserting the cannula into either end of the connector sleeve. Attach the other end of the connector sleeve to the patch cable.&#160; Apply enough pressure to secure the connection but be careful to not bend or break the optical fiber in the cannula. 
	NOTE: The fragile fiber optic cannula is covered with the protective cap whenever the laser is not in use.</li>
	<li>Set the LED driver to the continuous wave (CW) mode with the continuous adjustment knob at 0 and the current limit set to 1.2 A.</li>
</ol>
2. Photoconversion of Cells in the Colon
NOTE: Male and female mice were exposed to intracolonic 405 nm light 1 - 2 day(s) after their birth and were sacrificed prior to 1 week of age.
<ol>
	<li>Secure a dark location with an access to an electrical outlet. Plug the LED driver into the outlet. 
	NOTE: This procedure is performed in the minimal white light as a lengthy exposure of the mice to the ambient light may cause an unwanted photoconversion of cells. Red light is used as an alternative. 
	CAUTION:&#160;The following step involves the use of an animal anesthetic. The administration of the anesthetic takes place in a Class II hood or by using another appropriate scavenging system. The inhalation of the anesthetic may cause dizziness, drowsiness, or even unconsciousness.</li>
	<li>Anesthetize the mouse by exposure to 4 - 5% isoflurane in 100% O2 in an induction box. Verify proper depth of anesthesia by firmly pinching a hind paw (no response). Maintain anesthesia via a nose cone at approximately 2% isoflurane. Prevent handler exposure to isoflurane by use of an appropriate hood or anesthetic scavenging device.</li>
	<li>Position the anesthetized mouse for the intracolonic exposure by using 1 hand to scruff the back of the mouse gently with the index finger and thumb. Turn the mouse over to expose the abdomen. Loosen the grip if the mouse begins to lose its pink color or if its breathing rate begins to slow. Secure the tail of the mouse between the ring and little fingers.</li>
	<li>With the other hand, remove the protective cap from the cannula. While maintaining the long axis of the cannula parallel to the mouse&#39;s midline, gently insert the fiber optic cannula through the anus into the colon so that all 5 mm of the optical fiber are inside the mouse. 
	NOTE: This procedure is performed slowly and with care not to perforate the colonic wall. Minute wiggling and twisting of the cannula aids the advancement into the colon.</li>
	<li>Don the safety goggles and increase the laser current to the maximum value (all the way clockwise). Confirm the presence of UV light by looking at the translucent abdomen of the mouse. 
	NOTE: The LG3 laser safety goggles dramatically decrease visibility. Have a colleague (wearing protective equipment) assist in donning the goggles and turning on the laser.</li>
	<li>Expose the colon to the laser light for a total of 2 min. Withdraw the cannula approximately 1 mm every 30 s to maximize the luminal area exposed to the light. At the end of 2 min, turn the laser off.</li>
	<li>Slowly remove the cannula from the mouse. After recovering from anesthesia in an empty warm cage, return the mouse to the home cage.&#160;</li>
</ol>
3. Isolation of Intestinal Lymphocytes
<ol>
	<li>Prepare 1 L of the Hank&#39;s Balanced Salt Solution/calf serum (HBSS/CS) buffer, 200 mL of epithelial strip buffer, 500 mL of wash media, and 200 mL of ethylenediaminetetraacetic acid (EDTA)-free wash media. 
	NOTE: The reagent volumes are calculated for a sample size of 9.
	<ol>
		<li>Make the HBSS/CS buffer (HBSS; 5% CS) by adding 50 mL of CS to 950 mL of HBSS. Store it on ice.</li>
		<li>Prepare the epithelial strip buffer [HBSS; 5% CS; 1 mM dithiothreitol (DTT); 5 mM EDTA; 15 mM 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (pH 7.2 - 7.5)] by adding 10 mL of CS, 200 &#181;L of 1 M DTT, 2 mL of 0.5 M EDTA, and 3 mL of 1 M HEPES buffer to 185 mL of HBSS. Place the epithelial strip buffer in a 37 &#176;C water bath.</li>
		<li>Make the wash media (HBSS; 1% CS; 1 mM EDTA) by adding 5 mL of CS and 1 mL of 0.5 M EDTA to 494 mL of HBSS. Store it at room temperature.</li>
		<li>Prepare the EDTA-free wash media (HBSS; 1% CS; 15 mM HEPES) by adding 2 mL of CS and 3 mL of 1 M HEPES to 195 mL of HBSS. Store it at room temperature.</li>
		<li>Make a FACS buffer [1x phosphate buffered saline (PBS) (pH = 7.2); 2% CS] by adding 10 mL of CS to 490 mL of PBS. Store it on ice.</li>
	</ol>
	</li>
	<li>Prepare the tissue collection dish by placing a 70 &#181;m cell strainer inside a 60 x 10 mm culture dish. Add 10 mL of HBSS/CS buffer (HBSS; 5% CS buffer made in step 3.1.1 and stored on ice) to the dish and place it on the ice.</li>
	<li>Euthanize the mouse via an overdose of inhaled isoflurane using appropriate scavenging of anesthetic (an approximately 3 min exposure to &#62;4% isoflurane in the room air or in 100% O2). After apparent cessation of breathing and reflex movement, verify the absence of deep pain by firmly pinching a hind paw (using forceps is recommended). Ensure the euthanasia via a decapitation using either a pair of surgical scissors or a new straight razor. 
	NOTE: Cervical dislocation is not recommended for this step due to the size and age of the animal. These methods are consistent with the 2013 AVMA Guidelines on Euthanasia and approved by the MGH IACUC.</li>
	<li>Harvest the colon using clean forceps and scissors to open the abdomen. Reflect the intestines to 1 side of the mouse&#39;s abdomen to expose the colon. Remove the whole colon by first transecting it just distal (toward the anus) to the cecum. Then, while firmly grasping the colon with forceps, use scissors to cut through the urethra to the rectum. Cut the colon as close to the anus as possible. Harvest the spleen after removing the colon (see steps 4.1 - 4.2 below). 
	NOTE: While the photoconversion protocol only targets ~25% of the newborn&#39;s colon (5 mm of the fiber optic cannula), the entire colon is harvested to aid in the cell isolation.</li>
	<li>Clean the colon by using forceps to push the contents out of the colon and onto a dry paper towel. Homogenize the tissue by using razor blades to finely chop the colon into a paste on the cover of a culture dish and then transfer this paste to the cell strainer in the Petri dish.</li>
	<li>Add a clean 8 mm long magnetic stir bar to the strainer and place the dish on a 9-position magnetic stirrer plate. Set the magnetic stir bar speed to 800 rpm and incubate the cells for 5 min at room temperature. Discard the buffer and repeat the wash using 10 mL of HBSS/CS (the HBSS; 5% CS buffer made in step 3.1.1 and stored on ice). Discard the buffer at the end of the wash.</li>
	<li>Dissociate the epithelial cells by adding 10 mL of prewarmed epithelial strip buffer (the HBSS; 5% CS; 1mM DTT; 5mM EDTA; 15mM HEPES [pH 7.2 - 7.5] buffer that was made in step 3.1.2 and stored in a 37 &#176;C water bath) to the cell strainer and stir at 800 rpm for 30 min in a 37 &#176;C incubator. At the end of the incubation, transfer the buffer enriched in the epithelial fraction to a clean 15 mL tube. 
	NOTE: The epithelium and associated cells (intestinal epithelial cells and the intestinal epithelial lymphocytes) are released into the buffer, while the cells in the intact lamina propria remain on the cell strainer during this step11.
	<ol>
		<li>Centrifuge the enriched intestinal epithelial fraction (IELs) at 700 x g for 5 min at room temperature. Discard the supernatant and resuspend the cell pellet in 1 mL of FACS buffer. Store it on the ice for use in step 3.15.</li>
	</ol>
	</li>
	<li>Wash the lamina propria fraction within the cell strainer 2x with HBSS/CS. To do so, add 10 mL of HBSS/CS solution (HBSS; 5% CS buffer that was made in step 3.1.1 that has been stored on ice) to the cell strainer and stir it at 800 rpm for 5 min in a 37 &#176;C incubator. Discard the buffer after each wash.</li>
	<li>Wash the sample 2x in wash media. Add 10 mL of wash media (HBSS; 1% CS; 1 mM EDTA buffer that was made in step 3.1.3 and stored at room temperature) to the cell strainer and stir it at 800 rpm for 15 min in a 37 &#176;C incubator. Discard the buffer after each wash.
	<ol>
		<li>After using the wash media, store it on the ice for the use in step 3.14.</li>
	</ol>
	</li>
	<li>Wash the sample 2x with HBSS/CS. Add 10 mL of HBSS/CS buffer (HBSS; 5% CS buffer made in step 3.1.1 that has been stored on ice) to the cell strainer and stir it at 800 rpm for 5 min in a 37 &#176;C incubator. Discard the buffer after each wash.</li>
	<li>Incubate the tissue in EDTA-free wash buffer. Add 10 mL of EDTA-free wash buffer (HBSS; 1% CS; 15 mM HEPES buffer that was made in step 3.1.4 and stored at room temperature) to the cell strainer and incubate the plate for 10 min at room temperature without stirring. Discard the buffer at the end of this step.</li>
	<li>Wash the sample 2x with HBSS/CS and prepare the enzyme solution. Add 10 mL of HBSS/CS (HBSS; 5% CS buffer made in step 3.1.1 that has been stored on ice) buffer to the cell strainer and stir it at 800 rpm for 10 min in a 37 &#176;C incubator. Discard the buffer after each wash.
	<ol>
		<li>Prepare the enzyme solution (EDTA-free wash buffer; 0.1 mg/mL DNase I; 167 mg/mL collagenase) during the second wash period: add 9 mg of DNase I and 15 mg of lyophilized collagenase to 90 mL of EDTA-free wash buffer. Store the solution at room temperature.</li>
	</ol>
	</li>
	<li>Digest the lamina propria fraction contained in the cell strainer to obtain lamina propria lymphocytes (LPLs). Add 10 mL of the enzyme solution (the EDTA-free wash buffer; 0.1 mg/mL DNase I; 167 mg/mL collagenase buffer made in step 3.12.1 and stored at room temperature) to the cell strainer and stir it at 800 rpm for 45 min in a 37 &#176;C incubator. 
	NOTE: LPLs are released into the solution after the enzymatic digestion.
	<ol>
		<li>Collect the enzyme solution containing the cells and filter it through a new cell strainer into a 50 mL conical tube.</li>
	</ol>
	</li>
	<li>Add 20 mL of cold wash media (the HBSS; 1% CS; 1mM EDTA buffer made in step 3.1.3 and stored on ice in step 3.9.1) to the filtered cells to block the collagenase activity in the enzyme solution. Centrifuge the tube at 700 x g for 6 min at room temperature. Discard the supernatant and resuspend the pellet containing LPLs in 1 mL of FACS buffer.</li>
	<li>Combine the IEL (from step 3.7.1) and the LPL (from step 3.14) fractions and stain them with fluorescent-conjugated antibodies for the flow cytometric analysis (see step 5). 
	NOTE: IEL and LPL fractions may be stained separately if compartment information is desired.</li>
</ol>
4. Isolation of Lymphocytes from the Spleen
<ol>
	<li>Prepare the collection tube by adding 500 &#181;L of FACS buffer into a microcentrifuge tube. Store it on the ice.</li>
	<li>After removing the colon (step 3.4), harvest the whole spleen and place it in the collection tube. Store the tissue on ice.</li>
	<li>Using an electric tissue homogenizer, homogenize the spleen in a microcentrifuge tube for approximately 1 min.&#160;Centrifuge the sample at 300 x g [micro centrifuge] for 5 min at room temperature and aspirate off the supernatant.</li>
	<li>Perform the red blood cell lysis by resuspending the sample in 500 &#181;L of ammonium-chloride-potassium (ACK) lysis buffer to initiate cell death by osmotic lysis. Allow the sample to incubate 1 - 2 min at room temperature and then resuspend it in 725 &#181;L of FACS buffer.</li>
	<li>Centrifuge the sample at 300 x g [micro centrifuge] for 5 min at room temperature and aspirate off the supernatant. Resuspend the pellet in 500 &#181;L of FACS buffer and filter it through a 40 &#181;m cell strainer into a new microcentrifuge tube.&#160;Transfer the whole sample to a new microcentrifuge tube and stain it with fluorescent-conjugated antibodies for the flow cytometric analysis (see step 5).</li>
</ol>
5. Identification of Dendra-r+ Cells Using Flow Cytometry
<ol>
	<li>Centrifuge the IEL/LPL and spleen samples at 300 x g [micro centrifuge] for 5 min at room temperature and aspirate off the supernatant.</li>
	<li>Resuspend the pellets in 100 &#181;L of antibody staining mix. Prepare the antibody staining mix by adding 1 &#181;L of fluorescently-conjugated anti-CD45 antibody to 100 &#181;L of FACS buffer per sample. 
	NOTE: It is important to titrate the antibodies used for the flow cytometry prior to the experiment to determine the optimal concentration where binding saturation occurs.</li>
	<li>Incubate the samples for 35 min on ice (in a dark location).</li>
	<li>Resuspend the samples in 300 &#181;L of FACS buffer and centrifuge them at 300 x g [micro centrifuge] for 5 min at room temperature. Aspirate the supernatant and resuspend the samples in 325 &#181;L of FACS buffer. Analyze the samples on a flow cytometer.</li>
</ol>

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	<li>Maynard, C. L., Elson, C. O., Hatton, R. D., Weaver, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+of+the+intestinal+microbiota+and+immune+system.">Reciprocal interactions of the intestinal microbiota and immune system.</a> Nature. 489, 231-241 (2012).</li><li>Paun, A., Yau, C., Danska, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+the+Microbiome+on+Type+1+Diabetes.">The Influence of the Microbiome on Type 1 Diabetes.</a> Journal of Immunology. 198, 590-595 (2017).</li><li>Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+and+autoimmune+disease:+the+hosted+self.">Microbiota and autoimmune disease: the hosted self.</a> Cell Host Microbe. 10, 297-301 (2011).</li><li>Kollmann, T. R., Kampmann, B., Mazmanian, S. K., Marchant, A., Levy, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+Newborn+and+Young+Infant+from+Infectious+Diseases:+Lessons+from+Immune+Ontogeny.">Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from Immune Ontogeny.</a> Immunity. 46, 350-363 (2017).</li><li>Jain, N., Walker, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+and+host-microbial+crosstalk+in+postnatal+intestinal+immune+homeostasis.">Diet and host-microbial crosstalk in postnatal intestinal immune homeostasis.</a> Nature Reviews in Gastroenterology and Hepatology. 12, 14-25 (2015).</li><li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews in Immunology. 14, 667-685 (2014).</li><li>Macpherson, A. J., Uhr, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+protective+IgA+by+intestinal+dendritic+cells+carrying+commensal+bacteria.">Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria.</a> Science. 303, 1662-1665 (2004).</li><li>Mowat, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+basis+of+tolerance+and+immunity+to+intestinal+antigens.">Anatomical basis of tolerance and immunity to intestinal antigens.</a> Nature Reviews in Immunology. 3, 331-341 (2003).</li><li>Diehl, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+restricts+trafficking+of+bacteria+to+mesenteric+lymph+nodes+by+CX(3)CR1(hi)+cells.">Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.</a> Nature. 494 (3), 116-120 (2013).</li><li>Pham, A. H., McCaffery, J. M., Chan, D. 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The authors have nothing to disclose.

The identification and characterization of cells that interact with and are influenced by the microbiota in the colon are important and should facilitate understanding of how information from the mucosal microenvironment is relayed to the rest of the body. One method for studying gut-related cell migration requires the isolation of gut-associated cells followed by an adoptive transfer into recipient mice to determine their tissue-homing patterns and function12,13. This approach is limited, as only specific cell types that were transferred may be tracked. Further, artifacts resulting from the isolation of the cells from the tissues and the purification process may hinder the in vivo migratory process. More recently, an endoscopic photoconversion protocol to label intestinal cells using Kaede mice was reported14. This study found a broad trafficking of leukocytes to and from the intestine in adult mice.
Our report describes a method to label cells in the colon of newborn mice and track their migration to extra-intestinal sites such as the spleen. The PhAMexcised mouse ubiquitously expresses a photoconvertible green fluorescent Dendra2 protein that is irreversibly switched to red fluorescence upon its activation by UV light. The exposure of colons of newborn mice to 405 nm light labeled both CD45+ hematopoietic and CD45neg non-hematopoietic cells. CD45+Dendra2-red+ cells were detected in the spleen 3 to 5 days after the photoconversion, indicating the extra-intestinal migration of hematopoietic cells.
The rate-limiting step for this assay is the fraction of the colonic area that is exposed to 405 nm light. The fragility of the newborn colon and the presence of stool, albeit minimal at this early age, limit the length of the fiber optic cannula that can be inserted. In previous experiments, a longer cannula could not be successfully inserted more than 5 mm.&#160; Lubricants were&#160;avoided due to the risk of the variable light refraction of the laser beam. The outward movement of the cannula every 30 s helped maximize the colonic area exposed to the laser light. Importantly, a cannula with a large NA value (NA = 0.5) was used to generate a wide cone of light in the colon.
This protocol has been standardized for newborn (day 0 - 2) PhAMexcised pups. Given the changes in content and length of the colon with the age of the mice, the technique will have to be optimized accordingly when using older pups. Primarily, modifications to the total duration of the photoconversion and the length of the insertion of the fiber optic cannula will be necessary.
This approach to understanding the migratory behavior of cells in the colon is facilitated by flow cytometric analysis. Multi-parameter flow cytometry may be further used to identify and phenotype the migrant immune cell types within the CD45+ cell subset. Dendra2-r+ cells may also be sorted by FACS for downstream applications such as gene expression profiling or proteomics analyses. While directed to the understanding of hematopoietic cell subsets in the colon in this study, the method may be adapted to study cellular migration from additional tissue sites including the brain and the skin.

The mammalian gastrointestinal tract harbors hundreds of species of bacteria that exist in a symbiotic relationship with the host1. The immune cells present in the local milieu enforce a peaceful coexistence with these microbes and establish a protective barrier against pathogen invasions. Thus, bi-directional interactions between the immune cells and the microbiota are critical to establish a commensal community that educates the host immune system and sets the threshold for immune reactivity to pathogens. Changes in the microbial composition, or dysbiosis, can disturb the immune homeostasis and perturb regulatory circuits that restrain intestinal inflammations leading to immune-mediated diseases such as Type 1 Diabetes and IBD2,3.
The period immediately after birth is a unique developmental window during which the intestinal microbial communities begin to establish at the same time the immune system matures4. The postnatal microbiota is not stable, with shifts in the community composition occurring naturally and frequently5. The immune cells that interact with the microbiota reside in two distinct anatomical locations in the intestine - the lamina propria and the intestinal epithelium6. Numerous types of immune cells are present in the intestine, including lymphocytes (such as T cells, B cells, and innate lymphoid cells) as well as myeloid cells (which include dendritic cells, monocytes, and macrophages). These cells, also known as hematopoietic cells, perform a multitude of functions that preserve the intestinal barrier and maintain homeostasis.
In addition to their regulatory functions at intestinal sites, immune cells of the mucosa may also carry microbial messages to the extra-intestinal sites to regulate systemic immunity7,8,9. This is an area of growing research interest and highlights the need for methods to identify immune cells that migrate out of intestinal tissues in order to probe their function. The protocol reported here utilizes a commercially available mouse model in which a photoconvertible fluorescent protein is exploited to label cells. PhAMexcised mice ubiquitously express a green fluorescent Dendra2 protein that is irreversibly switched to red fluorescence upon activation by ultraviolet (UV) light10. Using a fiber optic cannula to deliver 405 nm light into the colon of newborn mice, we demonstrate that photoconverted hematopoietic cells, which have originated in or transited through the colon can be found in the spleen.

<table border="0" cellpadding="0" cellspacing="0" style="width:873px;" width="874">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Laser</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Light Emitting Diode (LED)</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M405FP1</td>
			<td style="width:247px;">CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">LED driver</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LEDD1B</td>
			<td style="width:247px;">Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Optogenetics patch cable</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M87L01</td>
			<td style="width:247px;">1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&#38;pn=M87L01#11454</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Fiber optic cannula</td>
			<td style="width:201px;">Doric lenses&#160;</td>
			<td style="width:167px;">MFC_480/500-0.5_5mm_ZF1.25_C45</td>
			<td style="width:247px;">5 mm long cannula with an outer diameter of 500 &#181;m and an inner diameter of 480 &#181;m. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Power supply</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">KPS101</td>
			<td style="width:247px;">Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">LG3 laser safety goggles&#160;</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LG3</td>
			<td style="width:247px;">Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&#38;pn=LG3#2523</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Red light&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>74327-10</td>
			<td style="width:247px;">15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Intestinal cell isolation</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Isoflurane</td>
			<td style="width:201px;">Patterson Veterinary</td>
			<td style="width:167px;">07-893-1389</td>
			<td style="width:247px;">CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&#160; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">1X HBSS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">14025076</td>
			<td style="width:247px;">Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&#38;searchTerm=14025076&#38;searchType=RAPID&#38;matchedCatNo=14025076</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Calf Serum</td>
			<td style="width:201px;">Hyclone AZM</td>
			<td style="width:167px;">197696</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">EDTA</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:167px;">15575020</td>
			<td style="width:247px;">0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">DTT</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10197777001</td>
			<td style="width:247px;">CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&#38;region=US</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">HEPES</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">15630080</td>
			<td style="width:247px;">1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Petri dish</td>
			<td style="width:201px;">Corning</td>
			<td style="width:167px;">353004</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&#38;searchTerm=08772F&#38;searchType=RAPID&#38;matchedCatNo=08772F</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">70 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">352350</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Micro magnetic stir bar</td>
			<td style="width:201px;">Fisherbrand</td>
			<td style="width:167px;">1451364</td>
			<td style="width:247px;">Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Magnetic stir plate</td>
			<td style="width:201px;">Corning Laboratory Stirrers</td>
			<td style="width:167px;">440826</td>
			<td style="width:247px;">https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&#38;persist=true&#38;ip=no&#38;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Collagenase</td>
			<td style="width:201px;">Roche</td>
			<td style="width:167px;">5401020001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&#38;region=US&#38;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">DNase I</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10104159001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&#38;region=US</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">1X PBS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">20012-027</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">Pipet aid</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">14387165</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">10 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357530</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">25 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357515</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">15 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339651</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">50 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339653</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Single cell suspension</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Eppendorf tubes</td>
			<td style="width:201px;">Seal-Rite</td>
			<td style="width:167px;">1615-5500</td>
			<td style="width:247px;">Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Tissue homogenizer&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td>K7495400000</td>
			<td style="width:247px;">Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Homogenizer tips&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td style="width:167px;">7495210590</td>
			<td style="width:247px;">Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">ACK lysing buffer</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">A10492-01</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">40 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">08-771-1</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Antibodies</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">BV786 anti-mouse CD45&#160;</td>
			<td style="width:201px;">BD</td>
			<td style="width:167px;">564225</td>
			<td style="width:247px;">Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Live/Dead</td>
			<td>Invitrogen</td>
			<td>L34962</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/L34962</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Other</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:247px;">Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades</td>
		</tr>
	</tbody>
</table>

in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

A fiber-optic cable was used to deliver 405 nm light into the colons of 2-day old PhAMexcised mice. In previous experiments, a 30 s exposure was determined to give a maximal photoconversion of colon cells with minimal cytotoxicity (Figure 1A). Therefore, sequential 30 s exposures of different segments of the colon were carried out as described in the protocol. Following the laser exposure, the mice were immediately euthanized, and the photoconversion of the cells was determined. Single-cell preparations of the total colonic tissue were stained with fluorescent-conjugated anti-CD45 antibody and the flow cytometric analysis was performed to detect photoconversion. Unconverted Dendra2-green (Dendra-g) protein has excitation and emission maxima at 490 and 553 nm respectively and can be detected in the FITC channel, whereas photoconverted Dendra2-red (Dendra-r) protein has excitation and emission maxima at 507 and 573 nm respectively and may be detected in the PE channel of a flow cytometer. Once irreversibly switched to its red form, Dendra2 is highly photostable and useful for long-term tracking applications.
No Dendra-r+ cells were detected in unlasered mice (Figure 1B Left). However, 405 nm light exposure resulted in distinct Dendra-r+ cell populations in both the CD45neg and CD45+ cell compartments (Figure 1B Right). To determine if the colonic photoconversion protocol resulted in the background labeling of cells at extra-intestinal sites, we harvested spleens from newly photoconverted mice (T = 0) and assayed for Dendra-r+ cells by flow cytometry. No Dendra-r+ cells were detected in the CD45neg or CD45+ cell compartments (Figure 1C), suggesting that the intra-colonic laser beam did not penetrate enough to photolabel cells in the spleen. Thus, any Dendra-r+ cells detected in the spleen after allowing time for the migration (T = 3d, 5d) should have originated from the colon.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57929/57929fig1.jpg" /> 
Figure 1: In vivo photoconversion of colonic tissue. Cells of the colon from 2-day old PhAMexcised pups were exposed to intra-colonic 405 nm light for 30 s as described. The photoconversion and labeling of the cells were determined immediately after the procedure (T = 0) by a flow cytometric analysis of single-cell preparations of the colonic tissue and spleen. (A) This representative flow cytometric dot plot shows the expression of CD45 (x-axis) and the staining of the live-dead dye (y-axis) in single-cell preparations from the colons of control and lasered mice. Panels B and C are representative flow cytometric dot plot overlays which show the expression of the Dendra2-green protein (Dendra2-g, horizontal-axis) and Dendra2-red protein (Dendra2-r, vertical-axis) in control-unexposed and exposed CD45+ (hematopoietic) and CD45neg (non-hematopoietic) cells in (B) the colon and&#160;(C) the spleen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57929/57929fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Data from a typical photoconversion experiment to identify migratory cells in the spleen are shown in Figure 2. The photoconversion of the cells in the colons of 2-day old PhAMexcised mice was performed as in Figure 1. Mice were euthanized 3 and 5 days after the exposure and the presence of Dendra-r+ cells in the spleen was determined by flow cytometry. There were no Dendra-r+ cells in the CD45neg cell subset, consistent with the view that the CD45neg cells are primarily epithelial and other structural cells of the intestine. Some CD45+ hematopoietic cells expressed Dendra-r, suggesting that they had been photoconverted in the colon and migrated to the spleen.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57929/57929fig2.jpg" /> 
Figure 2: CD45+ hematopoietic cells migrate from the colon to the spleen. Newborn (day 0 - 2) PhAMexcised mice were exposed to 405 nm light to photoconvert Dendra2 protein by intra-colonic exposure. The representative flow cytometry plots show the expression of the Dendra2-green protein (Dendra2-g, horizontal-axis) and Dendra2-red protein (Dendra2-r, vertical-axis) in CD45neg and CD45+ spleen cells of unexposed 5-days-old mice and exposed mice analyzed at 3 and 5 days (T = 3d, 5d) after the photoconversion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57929/57929fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

in-vivo-photolabeling-cells-colon-to-assess-migratory-potential

Nanyang Technological University

Research

JoVE Journal

Medicine

5.7K Views.  Massachusetts General Hospital. The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Video: In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...