Name: in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice
Uploaded: 2018-08-10T14:45:00.000+00:00
Duration: 8 min 39 s
Description: 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

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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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. 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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><tbody><tr><td>&lt;strong&gt;Laser&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Light Emitting Diode (LED)</td><td>THORLABS</td><td>M405FP1</td><td>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><td>LED driver</td><td>THORLABS</td><td>LEDD1B</td><td>Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td></tr><tr><td>Optogenetics patch cable</td><td>THORLABS</td><td>M87L01</td><td>1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&amp;amp;pn=M87L01#11454</td></tr><tr><td>Fiber optic cannula</td><td>Doric lenses&amp;nbsp;</td><td>MFC_480/500-0.5_5mm_ZF1.25_C45</td><td>5 mm long cannula with an outer diameter of 500 &amp;micro;m and an inner diameter of 480 &amp;micro;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><td>Power supply</td><td>THORLABS</td><td>KPS101</td><td>Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td></tr><tr><td>LG3 laser safety goggles&amp;nbsp;</td><td>THORLABS</td><td>LG3</td><td>Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&amp;amp;pn=LG3#2523</td></tr><tr><td>Red light&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>74327-10</td><td>15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td></tr><tr><td>&lt;strong&gt;Intestinal cell isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>07-893-1389</td><td>CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&amp;nbsp; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td></tr><tr><td>1X HBSS</td><td>Gibco</td><td>14025076</td><td>Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&amp;amp;searchTerm=14025076&amp;amp;searchType=RAPID&amp;amp;matchedCatNo=14025076</td></tr><tr><td>Calf Serum</td><td>Hyclone AZM</td><td>197696</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575020</td><td>0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td></tr><tr><td>DTT</td><td>Sigma</td><td>10197777001</td><td>CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&amp;amp;region=US</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td>1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td></tr><tr><td>Petri dish</td><td>Corning</td><td>353004</td><td>https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&amp;amp;searchTerm=08772F&amp;amp;searchType=RAPID&amp;amp;matchedCatNo=08772F</td></tr><tr><td>70 micron cell strainer</td><td>Falcon</td><td>352350</td><td>https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td></tr><tr><td>Micro magnetic stir bar</td><td>Fisherbrand</td><td>1451364</td><td>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><td>Magnetic stir plate</td><td>Corning Laboratory Stirrers</td><td>440826</td><td>https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&amp;amp;persist=true&amp;amp;ip=no&amp;amp;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td></tr><tr><td>Collagenase</td><td>Roche</td><td>5401020001</td><td>https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&amp;amp;region=US&amp;amp;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td></tr><tr><td>DNase I</td><td>Sigma</td><td>10104159001</td><td>https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&amp;amp;region=US</td></tr><tr><td>1X PBS</td><td>Gibco</td><td>20012-027</td><td>https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td></tr><tr><td>Pipet aid</td><td>Thermo Scientific</td><td>14387165</td><td>https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td></tr><tr><td>10 mL serological pipet</td><td>Falcon</td><td>357530</td><td>https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td></tr><tr><td>25 mL serological pipet</td><td>Falcon</td><td>357515</td><td>https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td></tr><tr><td>15 mL conical centrifuge tube</td><td>Thermo Scientific</td><td>339651</td><td>https://www.thermofisher.com/order/catalog/product/339650</td></tr><tr><td>50 mL conical centrifuge tube</td><td>Thermo Scientific</td><td>339653</td><td>https://www.thermofisher.com/order/catalog/product/339650</td></tr><tr><td>&lt;strong&gt;Single cell suspension&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eppendorf tubes</td><td>Seal-Rite</td><td>1615-5500</td><td>Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td></tr><tr><td>Tissue homogenizer&amp;nbsp;</td><td>Kimble</td><td>K7495400000</td><td>Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td></tr><tr><td>Homogenizer tips&amp;nbsp;</td><td>Kimble</td><td>7495210590</td><td>Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td></tr><tr><td>ACK lysing buffer</td><td>Gibco</td><td>A10492-01</td><td>https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td></tr><tr><td>40 micron cell strainer</td><td>Falcon</td><td>08-771-1</td><td>https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BV786 anti-mouse CD45&amp;nbsp;</td><td>BD</td><td>564225</td><td>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><td>Live/Dead</td><td>Invitrogen</td><td>L34962</td><td>https://www.thermofisher.com/order/catalog/product/L34962</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td>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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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 Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biology

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Immunology and Infection

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Cancer Research

Tracking Mouse Bone Marrow Monocytes In Vivo

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

Tracking Mouse Bone Marrow Monocytes In Vivo

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

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

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

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

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

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

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

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

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

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