imaging transgenic beta-cells in an immunodeficient mouse challenged with diabetic splenocytes

Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes

Warm up the body of the adult recipient, NOD-scid mice by using a heat lamp for 5 to 10 minutes to vasodilate the veins. Resuspend the splenocytes&#39; solution prior to each injection. For each mouse, draw 100 microliters of the pre-warmed splenocytes&#39; suspension into the syringe. Ensure that no air bubbles are present.
Next, place the mouse in the restraint device before capturing its tail with the non-dominant hand. Locate one of the two lateral tail veins. Gently rotate the tail if needed. Wipe the tail with a disinfection wipe containing 70% isopropyl alcohol.
Using the dominant hand, insert the needle at an acute angle into the central region of the tail. With the bevel of the needle facing up, slide the needle a few millimeters through the skin. Then, apply gentle pressure to the syringe to inject the splenocytes&#39; suspension. Do not allow the needle to move further in or out when injecting. Successful injections do not give rise to back pressure during injection, and are indicated by transparent or white blood flow immediately following injection.
After injection, gently release the needle out of the vein and apply light pressure to the tail with a disinfection wipe until the bleeding stops. Release the mouse from the restraint device and gently transfer it to a freshly prepared cage. At least 5 minutes before imaging, inject the mouse intraperitoneally with the appropriate dosage of D-luciferin solution, using a 1-milliliter syringe and a 26-gauge needle.
After logging in to the imaging software, in the control panel select Initialize. To autosave the imaging data, click on Acquisition followed by Auto-Save To, and create the appropriate folders in the computer. Once the instrument has finished initializing, set the exposure time to one minute.
Place the mouse on the imaging instrument in a prone position with its limbs splayed and guide its head into the nose cone. Flatten the mouse gently by pressing the center of its back with both hands, and then spreading the hands outward and apart. Then in the imaging software, select Acquire, and record any relevant details of the experiment.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Engineering and maintenance of NIT-1 cell lines
<ol>
	<li>Maintain NIT-1 cell lines, which share a genetic background with non-obese diabetic&#160;(NOD) mice, and 293FT cells in tissue culture-treated dishes in Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a 37 &#176;C incubator with 5% CO2.</li>
	<li>Grow the 293FT cells to 70%-80% confluence for transfection.</li>
	<li>Per 10 cm dish of 293FT cells, combine 10 &#181;g of pLenti-luciferase-blast (see&#160;Supplemental File 1), which expresses the firefly luciferase transgene (luc2) under the control of the constitutively active&#160;EF1&#945;&#160;promotor, 2 &#181;g each of the packaging plasmids pMD2.G, pMDLg/pRRE, and pRSV-Rev, 4 &#181;g of the envelope protein plasmid pCMV-VSV-G, and 60 &#181;g of linear polyethylenimine (PEI) in 1 mL of serum-free DMEM. Adjust the amounts based on the number of cells to be transfected.</li>
	<li>Leave the DNA, PEI, and DMEM at room temperature (RT) for 20 min, and then add them to the 293FT cells.</li>
	<li>Collect the medium containing the lentiviral particles 48 h post-transfection.</li>
	<li>Transduce the NIT-1 cells at ~80% confluency with 1 mL of medium containing lentiviral particles per 106&#160;cells for 48-72 h.</li>
	<li>Select the luciferase-expressing cells with 5 &#181;g/mL blasticidin for 48 h.</li>
	<li>Further, genetically modify the luciferase-expressing cells to suit the experimental question. Include a luciferase-expressing wild-type or non-targeting control cell line for comparison.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Examples of target gene modifications include clustered regularly interspaced short palindromic repeats (CRISPR) knockout, CRISPRa/i, shRNA knockdown, and overexpression.</li>
</ol>
2. Preparation of NIT-1 cells for transplant
<ol>
	<li>Grow the luciferase-expressing cells until they are 80%-90% confluent.</li>
	<li>Remove the growth medium and wash the cells with phosphate-buffered saline (PBS, 5 mL for a 10 cm dish).</li>
	<li>Add 1 mL of 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) to each dish for 2 min.</li>
	<li>Neutralize the trypsin with 5 mL of growth medium.</li>
	<li>Use a pipette to wash the cells off the dish and transfer them to a conical tube. 
	NOTE: Cells from multiple dishes of the same cell line can be combined.</li>
	<li>Count the cells using a stain such as trypan blue manually with a hemocytometer or automatically with an automatic cell counter.</li>
	<li>Centrifuge at 250 &#215;&#160;g&#160;for 5 min at RT and remove the supernatant with an aspirating pipette.</li>
	<li>Resuspend the cell pellet in PBS at a concentration of 5 &#215; 107&#160;cells/mL (107&#160;cells per cell line per mouse will be transplanted in a volume of 200 &#181;L).</li>
	<li>Keep the cells on ice (for up to 3 h) until transplantation.</li>
</ol>
3. Transplantation of NIT-1 cells into nonobese diabetic-severe combined immunodeficiency (NOD-scid) mice
<ol>
	<li>Anesthetize the recipient mice (8-10-week-old NOD-scid mice of the same sex as the mice used to isolate the splenocytes) by isoflurane inhalation in a knockdown chamber. Deliver 2.5% isoflurane into the chamber at a flow rate of 1.5 L/min and an evacuation rate of 9 L/min.&#160;</li>
	<li>Lubricate the animal&#39;s eyes with ophthalmic ointment to prevent drying.</li>
	<li>Using an electric shaver with a guard, remove the hair from the back (dorsal side) of the mice to expose the skin. The shaved transplant area will be approximately 1 in x 2 in. Return the shaved mice to the knockdown chamber until the transplant.</li>
	<li>Transfer the mice one at a time to a clean surface with an isoflurane nose cone. Gently guide the mouse&#39;s head into the nose cone. Use an isoflurane flow rate of 0.25 L/min to maintain the anesthesia during the transplant.</li>
	<li>Wipe the transplant area with an isopropyl alcohol prep pad to remove loose hair and disinfect the skin.</li>
	<li>Ensure the cells are fully resuspended before loading the syringe. Draw an excess volume (&#62;300 &#181;L) of cells into a 1 mL sterile syringe with a 26 G needle. Remove any bubbles; then return excess cells to the tube so that 300 &#181;L of cell suspension remains in the syringe.</li>
	<li>Using a pair of curved forceps held in the non-dominant hand, gently lift the skin on one side (left or right) of the mouse&#39;s back to allow for easier access to the subcutaneous space.</li>
	<li>Using the dominant hand, position the syringe parallel to the coronal and sagittal planes of the mouse&#39;s body. With the needle pointed toward the head of the mouse, insert the needle into the skin near the hindquarters and gently guide it into the subcutaneous space. Ensure that the entire needle remains under the skin and does not poke through.</li>
	<li>Adjust the forceps to gently hold the skin around the base of the needle. Slowly dispense a small amount of cell suspension (&#60;50 &#181;L) and confirm that a small bulge forms under the skin at the site of the injection. Continue injecting the cell suspension until 200 &#181;L has been delivered to the subcutaneous space.</li>
	<li>Holding the forceps in place and keeping the needle parallel to the mouse&#39;s body, slowly withdraw the needle. After the needle has been removed, hold the skin closed with forceps for a few seconds to prevent the cells from leaking out of the puncture wound.</li>
	<li>If a genetic modification is being evaluated, repeat the transplant on the opposite side of the mouse using a different syringe so that both mutant and control cells are injected into each mouse, with one cell line on the left and the other on the right. If only one cell line is being transplanted, inject the cells on one side only.</li>
	<li>Following the transplant, transfer each mouse to a fresh cage. Allow each mouse to fully recover from the anesthesia before adding additional mice to the cage. 
	NOTE: Mice are recovered when they resume normal activities and do not show signs of lethargy or impaired movement.</li>
	<li>Evaluate the health status of the mice daily for the duration of the experiment following the transplant. If the grafts form a large bulge or the mice become weak and lethargic, measure their blood glucose and provide 10% (w/w) sucrose water&#160;ad libitum&#160;to all the hypoglycemic mice. Follow all veterinary recommendations and euthanize any mice with continued poor body condition as per institutional guidelines. In this study, mice were euthanized by CO2&#160;inhalation followed by cervical dislocation.</li>
</ol>
4. Isolation and purification of autoreactive splenocytes
<ol>
	<li>Identify 10-16-week-old male or female NOD mice with recent-onset (less than 10 days) diabetes by using urine (or blood) glucose test strips. NOD mice are considered diabetic when they have urine/blood glucose &#8805;250 mg/dL on at least 2 consecutive days.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: For each NOD-scid mouse, 10 million splenocytes are needed; one spleen typically yields 50 million to 150 million splenocytes.&#160;The splenocytes were isolated from pathogen-free mice housed in a sentinal-monitored facility.</li>
	<li>Euthanize the appropriate number of diabetic NOD mice. 
	NOTE: In this study, the mice were euthanized by CO2&#160;inhalation followed by cervical dislocation to ensure death.</li>
	<li>With the mouse on its back, make a vertical incision approximately 2 in in length in the skin with surgical scissors. Open the right side of the mouse from the researcher&#39;s point of view and locate the bright red spleen. Gently cut the spleen away from the pink pancreas and transfer it to a sterile 10 cm Petri dish containing 5 mL of sterile PBS. Repeat the dissection with as many mice as needed. 
	NOTE: Spleens from multiple mice can be combined in one dish. Conduct the following steps using sterile reagents and equipment in a sterile hood.</li>
	<li>Mash the spleen(s) with the flat top of a sterile syringe plunger.</li>
	<li>Place a 40 &#181;m or 70 &#181;m strainer in a 50 mL conical tube and wash the strainer with 5 mL of PBS to prime it. Transfer the spleen(s) suspension into the strainer and continue gentle mashing through the strainer. Wash the dish with 10 mL of PBS and transfer the wash to the strainer. Continue mashing until the red color is gone from the strainer.</li>
	<li>Discard the strainer and spin the tube at 500 &#215;&#160;g&#160;for 5 min at RT. Remove the supernatant with an aspirating pipette.</li>
	<li>Resuspend the cell pellet in 5 mL (for up to three spleens) of ACK lysis buffer prewarmed to RT and lyse the red blood cells for 4 min. Increase the volume by 1-2 mL per spleen if additional spleens are needed.</li>
	<li>Stop the reaction with 5 mL of NIT-1 cell media (described above) per 5 mL of lysis buffer. Pass the cell suspension through a fresh strainer to remove clumps. Discard the strainer, and spin at 500 &#215;&#160;g&#160;for 5 min at RT.</li>
	<li>Resuspend the cell pellet in 20 mL of PBS, and count the cells. Spin at 500 &#215;&#160;g&#160;for 5 min at RT. Resuspend the cells in sterile PBS at a concentration of 1 &#215; 108&#160;cells/mL (the injection volume is 0.1 mL/mouse) in a 1.5 mL safe-lock reaction tube.</li>
	<li>Store the cells on ice (for not more than 1 h) or keep the cells at RT, and proceed to the tail vein injection immediately.</li>
</ol>
5. Intravenous injection of diabetic splenocytes&#160;via&#160;the lateral tail vein
<ol>
	<li>Warm up the body of the adult recipient NOD-scid mice (e.g., by using a heat lamp for ~5-10 min) to vasodilate the veins (this helps for visualization and injection into the vein). 
	NOTE: To avoid the overheating of the mice, do not exceed this time. Always monitor the health status. If the mice appear exhausted or immobile, shut off the heat lamp immediately.</li>
	<li>Warm up the cell suspension. Prepare a sterile syringe (0.3-1.0 mL) with a sterile needle (27-30 G, 0.3 mm/0.5 in or smaller), or use a sterile 0.5 mL insulin syringe with a 0.3 mm (0.5 in) needle. Always keep the needle sterile and use a sterile tray if the syringe must be put down between injections.</li>
	<li>Resuspend the splenocyte solution prior to each injection. For each mouse, draw 100 &#181;L of the prewarmed and mixed splenocyte suspension into the syringe. Make sure that no air bubbles are present in the syringe or in the suspension. 
	NOTE: Make sure to have all the equipment and supplies prepared (alcohol wipes for disinfection, a syringe loaded with the splenocytes to be injected, and a fresh cage for separating injected mice) before placing the mouse in the restraint device.</li>
	<li>Place the mouse in the restraint device. Capture the tail with the non-dominant hand and locate one of the two lateral tail veins. Gently rotate the tail if needed. Wipe the tail with a disinfection wipe (70% isopropyl alcohol) to clean the skin and increase the visibility of the vein.</li>
	<li>Insert the needle at an acute angle into the central region of the tail with the dominant hand. With the bevel of the needle facing up, slide the needle a few millimeters through the skin.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Make sure the needle is parallel to the vein and placed just slightly under the skin. Be prepared for sudden movements of the mouse/tail directly after penetrating the skin/vessel wall. A successful insertion of the needle should feel like a &#34;smooth slide&#34; into the vein. If another attempt is needed, move further up the tail toward the body.</li>
	<li>Apply gentle pressure to the syringe to inject the splenocyte suspension. Do not allow the needle to move further in or out when injecting. Successful injections are smooth without feeling any back pressure during injection and are indicated by a transparent/white blood flow immediately following injection.</li>
	<li>Gently release the needle out of the vein and apply light pressure to the tail with a disinfection wipe until the bleeding stops. Release the mouse from the restraint device, and gently transfer it to a freshly prepared cage.&#160;&#160; 
	NOTE: Inject two to three control mice that do not receive a NIT-1 cell transplant with splenocytes to confirm the potential of the autoreactive splenocytes to induce diabetes, which takes approximately 2-4 weeks post injection.</li>
</ol>
6.&#160;In vivo&#160;bioluminescent imaging of NIT-1 grafts
NOTE: Image the grafts one to two times per week. On the day of the transplant, wait at least 2 h after the transplant to allow the grafts to settle and ensure stable luciferase expression. If time is a limiting factor, the initial measurement may be taken on Day 1 instead. A recommended initial imaging schedule is Day 0 or Day 1 post injection, Day 5, Day 10, Day 14, Day 18, and Day 25. Adjust the schedule, however, based on the progress of autoimmunity as judged by the loss of bioluminescent signal.
<ol>
	<li>Prepare a 15 mg/mL D-luciferin solution in Dulbecco&#39;s PBS. Agitate at RT to dissolve, and sterile filter (0.22 &#181;m). Store 1 mL aliquots at &#8722;20 &#176;C, and thaw as needed. 
	NOTE: The aliquots can be refrozen.</li>
	<li>At least 5 min prior to imaging, inject the mice intraperitoneally using a 1 mL syringe and a 26 G needle with D-luciferin solution at a dose of 150 mg/kg.&#160; 
	NOTE: Use a fresh syringe and needle for each mouse.&#160;</li>
	<li>Anesthetize the mice by isoflurane inhalation according to institutional guidelines. The recommended conditions are 2.5% isoflurane with a flow rate of 1.5 L/min into the knockdown chamber and an evacuation rate of 9 L/min.</li>
	<li>Lubricate the animal&#39;s eyes with ophthalmic ointment to prevent drying.</li>
	<li>Open the software associated with the imaging instrument. Create a&#160;new user&#160;and/or&#160;login. On the&#160;control panel&#160;on the bottom right, select&#160;Initialize&#160;(Figure 1A). To&#160;auto-save&#160;the imaging data, create the appropriate&#160;folders&#160;on the computer, and then select&#160;Acquisition | Auto-save to&#8230;&#160;(Figure 1A). Once the instrument has finished initializing, set the&#160;exposure time&#160;to&#160;1 min.&#160;&#160;&#160;&#160; 
	NOTE: The complete imaging parameters are shown in&#160;Figure 1B.</li>
	<li>Transfer the mice one at a time from the knockdown chamber to the imaging instrument. Place the mouse on its stomach with its limbs splayed, and gently guide its head into the nose cone. An isoflurane flow rate of 0.25 L/min delivered&#160;via&#160;a nose cone is appropriate to maintain anesthesia during imaging. Gently flatten the mouse by pressing the center of its back with both hands and then spreading the hands outward and apart.</li>
	<li>Select&#160;Acquire&#160;(Figure 1B). Record any relevant details of the experiment in the pop-up window (Figure 1C).&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: See&#160;Figure 2A&#160;for representative images of three 8-week-old female NOD-scid mice transplanted with two grafts at various time points.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin, 0.53 mM EDTA</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td>Fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads, 70% Isopropyl alcohol</td>
			<td>Amazon/Ever Ready First Aid</td>
			<td>B08NWF31DX</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 5ml Syringe Luer-Lok Tip</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 26G x 5/8 (0.45 mm x 16 mm) Sub-Q</td>
			<td>BD</td>
			<td>305115</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL TB Syringe Slip Tip</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin S HCl</td>
			<td>Corning</td>
			<td>&#160;30-100-RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer premium SureStrain, 70 &#181;m, sterile</td>
			<td>Southern Labware</td>
			<td>C4070</td>
			<td>Or use similar sterile strainer with 40-70um pore size</td>
		</tr>
		<tr>
			<td>CellDrop automated cell counter</td>
			<td>Denovix</td>
			<td>CellDrop BF-PAYG</td>
			<td>Or use similar cell counter device</td>
		</tr>
		<tr>
			<td>Corning 100 mL Penicillin-Streptomycin Solution, 100x</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Aspirating Pipets, Polystyrene, Sterile, Capacity=2 mL</td>
			<td>VWR</td>
			<td>414004-265</td>
			<td>Or use similar aspirating pipette</td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt , Molecular Biology Grade, Powder, &#62;99%</td>
			<td>Goldbio</td>
			<td>LUCK-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate, no glutamine</td>
			<td>Gibco</td>
			<td>10313039</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon BD tubes, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps premium for tissues, 1 x 2 teeth 5 in, German Steel</td>
			<td>Fisher Scientific</td>
			<td>13-820-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose urine test strip</td>
			<td>California Pet Pharmacy</td>
			<td>u-tsg100</td>
			<td>Or use similar test strip for glucose measurments in urine/blood</td>
		</tr>
		<tr>
			<td>GlutaMAX&#8211;1 (100x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared heating lamp</td>
			<td>Cole Parmer</td>
			<td>03057-00</td>
			<td>Or use similar infrared lamp&#160;</td>
		</tr>
		<tr>
			<td>Insulin syringe 0.5 mL, U-100 29 G 0.5 in</td>
			<td>Becton Dickinson</td>
			<td>309306</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Piramal Critical Care</td>
			<td>6679401725</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum in vivo imaging system</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Instrument for non-invasively collecting bioluminescent images of transplanted cells</td>
		</tr>
		<tr>
			<td>Living Image Analysis Software</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td>Software for collecting and quantifying bioluminescent signal</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes seal-rite, 1.5 mL</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td>Or use similar sterile microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>NIT-1</td>
			<td>ATCC</td>
			<td>CRL-2055</td>
			<td>Pancreatic beta-celll line derived from NOD/Lt mice</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid/J</td>
			<td>The Jackson Laboratory</td>
			<td>1303</td>
			<td>Mice homozygous for the severe combined immune deficiency spontaneous mutation Prkdcscid, commonly referred to as scid, are characterized by an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment.</td>
		</tr>
		<tr>
			<td>NOD/ShiLtJ</td>
			<td>The Jackson Laboratory</td>
			<td>1976</td>
			<td>The NOD/ShiLtJ strain of mice (commonly called NOD) is a polygenic model for autoimmune type 1 diabetes</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010031</td>
			<td>No calcium, no magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>8454</td>
			<td></td>
		</tr>
		<tr>
			<td>pLenti-luciferase-blast</td>
			<td>Made in-house</td>
			<td>Plasmid available upon request</td>
			<td>See Supplemental File 1</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, Linear, MW 25,000, Transfection Grade (PEI 25K)</td>
			<td>Fisher Scientific</td>
			<td>NC1014320</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer for rodents, broome-style round 1 in</td>
			<td>Fisher Scientific</td>
			<td>01-288-32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, sharp-pointed</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
			<td>Or use other scissors made of surgical-grade stainless steel</td>
		</tr>
		<tr>
			<td>Tissue-culture treated culture dishes</td>
			<td>Millipore Sigma</td>
			<td>CLS430167-20EA</td>
			<td>Or use other sterile cell culture-treated Petri dishes</td>
		</tr>
		<tr>
			<td>Tweezers/Forceps, fine precision medium tipped</td>
			<td>Fisher Scientific</td>
			<td>12-000-157</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Stewart, T. et al., <a href="https://app.jove.com/v/64836">Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates an imaging-based method to visualize transplanted luciferase-expressing beta cells into a non-obese diabetic immunodeficient mouse. The introduction of splenocytes from a diabetic mouse leads to progressive beta cell destruction, visually confirmed by declining luminescence after substrate introduction.

<img alt="Figure 1" src="/files/ftp_upload/22278/22278_Figure1.jpg" /> 
Figure 1: Screenshots of the software commands for imaging bioluminescent grafts. (A) Prior to imaging, select Initialize to prepare the instrument. The images may be auto-saved to a folder of choice by selecting Acquisition | Auto-save to&#8230; (B) Overview of the imaging parameters. Once the mouse has been pos...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a restrained NOD immunodeficient mouse that lacks immune cells and is prone to diabetes.
This mouse is pre-injected with transgenic pancreatic beta-cells that produce insulin and express luciferase enzymes.
Take a syringe containing pre-treated splenocytes derived from a diabetic mouse. The splenocytes include autoreactive T cells with the ability to recognize the insulin peptides.
Administer these splenocytes into the mouse&#39;s tail vein.
The splenocytes circulate and reach the pancreas, where the autoreactive T cells recognize the insulin peptides on the beta cells and become activated.
These activated cells release cytotoxic molecules, initiating beta-cell destruction via autoreactivity.
Next, inject a luciferase substrate into the peritoneum of the mouse. The substrate diffuses in this region and enters the beta cells.
Within the cells, the substrate interacts with the luciferase enzymes, producing luminescence.
Using a bioluminescent imaging system, capture images at regular intervals
A gradual decrease in luminescence indicates the destruction of transgenic beta-cells due to autoreactivity.

17 vistas. Obtención de imágenes de células beta transgénicas en un ratón inmunodeficiente desafiado con esplenocitos diabéticos

Video: Obtención de imágenes de células beta transgénicas en un ratón inmunodeficiente desafiado con esplenocitos diabéticos - Experimento

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

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

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

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

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

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

Investigación

JoVE Journal

Inmunología e Infección

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

Expresión transitoria en remolacha roja de una vacuna candidata biofarmacéutica para la Diabetes tipo 1

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

Bioingeniería

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Generación de alta eficiencia de células T citotóxicas de ratón primario específicas de antígenos para pruebas funcionales en un modelo de diabetes autoinmune

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes

Transcripción

Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes