Name: imaging cd4 t cell interstitial migration in the inflamed dermis
Uploaded: 2016-03-25T14:00:00
Description: Watch this Scientific Journal Video about Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis at JoVE.com

De auteurs danken de Universiteit van Rochester Multiphoton Microscope Core faciliteit voor hulp bij het live-imaging. Ondersteund door NIH AI072690 en AI02851 naar DJF; AI114036 naar AG en AI089079 te MGO.

Alle procedures waarbij muizen werden goedgekeurd door de Institutional Animal Care en gebruik Comite van de Universiteit van Rochester, en in strikte overeenstemming met de Animal Welfare Act en de Public Health Service Beleid inzake Humane zorg en het gebruik van proefdieren beheerd door de National Institutes uitgevoerd van Volksgezondheid, Office of Laboratory Animal Welfare. 1. Bereiding van effector CD4 T-cellen LET OP: BALB / c TCR-transgene DO11.10 muizen d...

Betekenis Hier presenteren wij u een protocol voor 4D visualisatie overgedragen, antigeen-specifieke effector Th1 cellen in het intacte muizenoor dermis. Deze werkwijze biedt voordelen ten opzichte van sommige van de huidige beeldvormingsmodaliteiten om verschillende redenen. Door de beeldvorming van de ventrale oor dermis, zijn we in staat om ontharing die nodig is voor de beeldvorming protocollen waarbij andere huid websites af te zien. Hoewel ontharingsmiddelen algemeen mild, ble...

The effector function of CD4 T cells is critically dependent on their ability to rapidly enter and traverse a wide variety of peripheral tissues to survey for damage, locate foci of infection, or cause pathology from chronic infection or autoimmunity. While the processes of homing to inflamed sites1-4 and extravasation5-7 from the vasculature into tissues have been well-characterized, the factors that drive and regulate the interstitial motility of T cells remain undefined. The migration of T cells in complex 3D environments has been studied in vitro through the use of artificial matrices8-10 or microfluidic devices11,12, but these fail to recapitulate the complex and dynamic environment of an in vivo system. It is only recently, with the advent of high-resolution multi-color intravital imaging that it has become possible to study the dynamic behavior of immune cells in situ, allowing for a better understanding of intact immune responses.
Over a decade ago, several influential studies were published that first utilized multiphoton microscopy to address immunological questions. Early studies focused on the behavior of immune cells within explanted lymphoid organs13-16, which were soon followed by techniques to image exposed lymph nodes in anesthetized mice17. Imaging allowed for new fundamental observations about the stages of lymph node priming of T cells18, the mechanisms by which T cells migrate in secondary lymphoid organs19, T cell interactions with other immune cells20,21, and dynamic T cell positioning within the lymph node22. Although many early studies focused on lymph node dynamics, intravital imaging has been since been utilized to image the immune response in many peripheral tissues, including the brain23-25, liver26, lung27, and skin28-30. 
The mouse ear dermis is particularly well poised for imaging, due to the thinness of ear skin, a relative lack of hair, and the ease with which it can be isolated from respiratory movements31. Indeed, the ear dermis has been used to image the interstitial behavior of dendritic cells32,33, T cells28,29,34,35, and neutrophils36,37, and is a well-established site for studying dermal inflammation. Increasingly, non-invasive procedures have been replacing surgical preparations of the skin, including split dermis38,39, flank39,40, or dorsal skin flap window39,41 models, that can induce changes to the local inflammatory milieu. The use of transferred, in vitro-primed, antigen-specific CD4 effector T cells allows for the study of a homogenous population of cells in the context of a dermal inflammatory response30. Here we describe a non-invasive imaging procedure that allows for the visualization of antigen-specific effector CD4 T cells in the dermal interstitium of the inflamed mouse ear, and the ability to manipulate these cells in real-time by introducing blocking antibodies through a venous catheter. We show that this model is effective for tracking the movement of CD4 T cells in the dermis and for querying the mechanisms that govern this motility.

<table border="0" cellpadding="0" cellspacing="0" width="1060">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">BALB/c mice</td>
			<td style="width:145px;">Jackson Laboratories</td>
			<td style="width:196px;">000651</td>
			<td style="width:472px;">Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DO11.10 mice</td>
			<td>Jackson Laboratories</td>
			<td>003303</td>
			<td>Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HBSS</td>
			<td>Fisher</td>
			<td>10-013-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Newborn Calf Serum (NCS)</td>
			<td>Thermo/HyClone</td>
			<td>SH30118.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Guinea Pig Complement</td>
			<td>Cedarlane</td>
			<td>CL-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD8 antibody</td>
			<td>ATCC</td>
			<td>3.155 (ATCC TIB-211)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-MHC Class II antibody</td>
			<td>ATCC</td>
			<td>M5/114.15.2 (ATCC TIB-120)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD24 antibody</td>
			<td>ATCC</td>
			<td>J11d.2 (ATCC TIB-183)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-Thy1.2 antibody</td>
			<td>ATCC</td>
			<td>J1j.10 (ATCC TIB-184)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ficoll (Fico/Lite-LM)</td>
			<td>Atlanta Biologicals</td>
			<td>I40650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PBS</td>
			<td>Fisher</td>
			<td>21-040-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">EDTA</td>
			<td>Fisher</td>
			<td>15323591</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">biotinylated anti-CD62L antibody (clone MEL-14)</td>
			<td>BD</td>
			<td>553149</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">streptavidin magnetic separation beads</td>
			<td>Miltenyi</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MACS LS Separation Column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant human IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-4</td>
			<td>Peprotech</td>
			<td>214-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-12</td>
			<td>Peprotech</td>
			<td>210-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IFNg antibody (clone XMG 1.2)</td>
			<td>eBioscience</td>
			<td>16-7311-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IL-4 antibody (clone 11b11)</td>
			<td>eBioscience</td>
			<td>16-7041-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td>VWR</td>
			<td>45000-412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>15303641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">L-glutamine</td>
			<td>Fisher</td>
			<td>15323671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ovalbumin peptide</td>
			<td>Biopeptide</td>
			<td></td>
			<td>ISQAVHAAHAEINEAGR-OH peptide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Calf Serum (FCS)</td>
			<td>Thermo/HyClone</td>
			<td>SV30014.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">24-well culture plate</td>
			<td>LPS</td>
			<td>3526</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CFSE</td>
			<td>Life Technologies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 1ml</td>
			<td>BD</td>
			<td>329420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 300&#956;l</td>
			<td>BD</td>
			<td>309301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">27 G1/2 TB syringes, 1ml</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">30 G1/2 needles</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PE-10 medical tubing</td>
			<td>BD</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">cyanoacrylate veterinary adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">heating plate</td>
			<td>WPI</td>
			<td>61830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Heating plate controller</td>
			<td>WPI</td>
			<td>ATC-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Water blanket controller</td>
			<td>Gaymar</td>
			<td>TP500</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">water blanket</td>
			<td>Kent Scientific</td>
			<td>TP3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane vaporizer</td>
			<td>LEI Medical</td>
			<td>Isotec 4</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane</td>
			<td>Henry Schein</td>
			<td></td>
			<td>Ordered through Veterinary staff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">microcentrifuge tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">medical tape</td>
			<td>3M</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane nosecone</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">imaging platform</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">curved forceps</td>
			<td>WPI</td>
			<td>15915-G</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">glass coverslips</td>
			<td>VWR</td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">high vacuum grease</td>
			<td>Fisher</td>
			<td>146355D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cotton swabs</td>
			<td></td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">delicate task wipes</td>
			<td>Fisher</td>
			<td>34155</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Olympus Fluoview 1000 AOM-MPM upright microscope with Spectra-Physics MaiTai HP DeepSee Ti:Sa laser</td>
			<td>Olympus</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">optical table with vibration control</td>
			<td>Newport</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">25x NA 1.05 water immersion objective for multiphoton imaging</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">objective heater</td>
			<td>Bioptechs</td>
			<td>PN 150815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Detection filter cube</td>
			<td>Olympus</td>
			<td>FV10-MRVGR/XR</td>
			<td>Proprietary cube, can be approximated from individual filters/dichroics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;1 antibody (clone hMb1-1)</td>
			<td>eBioscience</td>
			<td>16-0291-85</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;3 antibody (clone 2C9.G3)</td>
			<td>eBioscience</td>
			<td>16-0611-82</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Texas Red Dextran (70,000 MW)</td>
			<td>Life Technologies</td>
			<td>D-1830</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cd4 t cell interstitial migration in the inflamed dermis

Het vermogen van CD4 T-cellen om effectorfuncties uitvoeren is afhankelijk van de snelle en efficiënte migratie van deze cellen in ontstoken perifere weefsels door middel van een vooralsnog ongedefinieerde mechanisme. De toepassing van multifoton microscopie voor de studie van het immuunsysteem een ​​werktuig om de dynamiek van immuunresponsen in intacte weefsels te meten. Hier presenteren we een protocol voor niet-invasieve beeldvorming van intravitale multifoton CD4 T-cellen in de ontstoken muisoor dermis. Gebruik van een aangepaste imaging platform en een veneuze katheter maakt de visualisatie van CD4 T-cel dynamica in de huid interstitium, met de mogelijkheid om deze cellen in real-time uitlezen via de toevoeging van blokkerende antilichamen tegen belangrijke moleculaire componenten betrokken bij motiliteit. Dit biedt voordelen ten opzichte van in vitro modellen en chirurgisch invasieve beeldvorming procedures. Het begrijpen van de paden die worden gebruikt door CD4 T-cellen voor de beweeglijkheid kan uiteindelijk geven inzicht in de basic functie van CD4 T-cellen en de pathogenese van zowel auto-immuunziekten en pathologie van chronische infecties.

Het vermogen om immuunreacties in situ te bestuderen zonder dat het immuunsysteem milieu essentieel bestuderen real-time interactie van T-effectorcellen met een ontstoken weefsel. Beeldvorming van het intacte oor dermis van dit protocol geschetst in figuur 1A en B, maakt de visualisatie overgedragen fluorescent gelabelde T effectorcellen in de huid interstitium. Dit maakt zowel hoge-resolutie (figuur 1C) en time-lapse (f...

Watch this Scientific Journal Video about Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis at JoVE.com

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Beeldvorming CD4 T Cell interstitiële Migratie in de ontstoken Dermis

De mechanismen die de interstitiële beweeglijkheid van CD4-effector-T-cellen regelen op plaatsen van ontsteking zijn relatief onbekend. Wij presenteren een niet-invasieve benadering visualiseren en manipuleren in vitro geprimed CD4 T-cellen in het ontstoken oor dermis, waardoor studie van het dynamisch gedrag van deze cellen in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

The overall goal of this imaging protocol is to visualize CD4 positive T Cell dynamics in the inflamed skin. This method can help answer key questions in the field of immunology, such as, what are the mechanisms that underlie immune cell motility, and function in inflamed tissues. The main advantage of this technique, is that it provides a minimally invasive way to study CD4 T cell dynamic behavior In Situ.Visual demonstration of this method is critical, as the steps to prepare the ear for imaging are difficult to learn, and proper positioning of the ear is necessary to capture high quality images. I'm Alison Gaylo, a graduate student, and I will be doing the demonstration of this protocol. Begin by aspirating 300 microliters of fluorescently labeled effector T cell suspension per mouse into a syringe equipped with a 27 and a half gauge needle.Then turn the syringe needle side up, and gently flick the shaft of the syringe, moving the plunger up and down as necessary to remove any bubbles. When the cells are ready, transfer the mice into a clean cage, and place them under a heat lamp, until the tail veins are vasodilated. Then transfer the first mouse into the restrainer, and wipe the tail with an alcohol swab.After identifying a lateral tail vein, slowly inject 200 microliters of cells into the vein. When all of the cells have been transferred, draw 50 microliters of complete fluorescens emulsion into a 28 and a half gauge insulin syringe and press down firmly on the plunger to remove any large air bubbles. Then place a thimble on the left index finger, and carefully grasp the mouse's ear between the left thumb and thimble, with the ventral ear facing up.Then slide the needle, bevel side up, into the dermis in the outer third of the pinna, and slightly off center, and slowly inject ten microliters of emulsion into the tissue. After injecting all of the animals, monitor the mice until they are fully recovered. Three days following the immunization, vasodilate the tail veins of the injected animals as just demonstrated.After anesthetizing, immobilize the tail of the first mouse with a pair of forceps. Wipe the tail with an alcohol swab, then carefully slide a 30 and a half gauge needle catheter into a lateral tail vein, checking for patency by gentle pressure to the syringe plunger. When the catheter is in place, apply one to two drops of cyanoacrylate tissue adhesive to the injection site, and allow the adhesive to dry for approximately 30 seconds.Then use scissors to carefully trim the whiskers and the hair from the back and sides of the ear. Next, use a cotton swab to moisten the inner surface of the ear with PBS. Then rotate the mouse, and flatten the injected ear onto a 24 by 50 millimeter number one point five glass cover slip, until it is flush with the glass.Blot away the excess PBS. Then use two pairs of curved forceps to grasp an approximately 20 millimeter long piece of fabric tape lengthwise at the top corners, and place the bottom of the tape onto the cover slip at the top of the mouse's ear. Roll the tape over the rest of the ear, pushing the excess hair out of the way with the forceps as necessary, and use a dry cotton swab to gently press the tape around the ear to ensure a tight seal, taking care not to press on the ear itself.It is essential that the ear is taped properly to the cover slip with a good seal on both sides and with minimal air bubbles, as this will affect the quality of the images that can be obtained. Now tape a fluorescent microscope imaging platform to a 37 degree Celsius heating block, and apply vacuum grease to both sides of the ear area of the platform. Rotate the mouse to place the cover slip onto the platform, taking care to align the ear in the center of the felt.When the ear is in place, snap the isoflurane nose cone into the holder on the platform, ensuring that the cone is secure, and completely covering the mouse's nose. Then use a cotton swab to firmly, but carefully, press the cover slip onto the imaging platform, spreading the vacuum grease under the cover slip. Wrap two 20 millimeter pieces and two longer pieces of tape around the upper portion of the platform to affix the cover slip to the platform.Next, move the mouse to the microscope stage. Secure the platform with more tape, and pipe a double layer of vacuum grease onto the cover slip around the ear. Then wrap a water filled heating blanket around the mouse, and fill the vacuum grease reservoir with 37 degrees Celsius distilled water.For In Vivo time lapse imaging, begin by positioning the objective over the center of the ear, and lower the objective until it just contacts the surface of the water in the reservoir. Next, using an external light source, slowly lower the objective until the surface of the ear comes into focus. Then, lower the inner and outer curtains around the microscope stage, and adjust the laser wave length to 900 nanometers in the MP laser controller window, the laser power in the acquisition setting laser window, and the PMT voltages in the image acquisition control window.In the acquisition setting size and mode windows, set the microscope to five twelve by five twelve pixel resolution, with a two microsecond per pixel dwell time, and select XY repeat to activate a live imaging mode for scanning through the tissue. Once an appropriate imaging field has been located, locate the highest cell in the dermis, and click the set zero button to set the Z position to zero. Scroll down in the Z direction to measure the extent of the cell depth.Then set the starting and end positions in the acquisition setting microscope window, and adjust the instrument PMT voltages and laser power in the bright Z window to optimize the visualization of the cells throughout the depth of the imaging field. Check the depth and time buttons, and set a common filter to scan the image three times per line in the image acquisition control filter mode window. Adjust the Z slice depth in the image acquisition microscope window so that it takes approximately one minute to capture a complete stack as noted in the time view window.Then, to assess the stability of the tissue, set the number of repeats to five in the acquisition setting time scan window, and click the scan button to capture a five minute time lapse image of the area. If the tissue is stable after five minutes, set the number of repeats to between 30 and 45 in the acquisition setting time scan window, and collect a 30 to 45 minute time lapse image, monitoring for any minor tissue drift as the image is collected. Save the pre-antibody image in the appropriate format, and then draw the antibody mixture into a one milliliter tuberculin syringe, taking care to remove all of the air bubbles.Gently press the plunger so that the antibody solution forms a droplet at the end of the needle, and lift the curtains around the stage to locate the catheter. Replace the PBS syringe with the syringe of antibody, and then slowly inject the antibodies into the catheter. When the antibodies have been successfully injected, re-lower the curtains and note the time of injection.Then immediately start collecting a new 20 to 40 minute imaging sequence, in the same location, using the same instrument settings as the pre-antibody image. Finally, save the post-antibody image file in the appropriate format, and carefully remove the mouse from the microscope stage, monitoring the animal until it is fully recovered. Imaging of the intact ear dermis by this protocol facilitates the acquisition of high resolution and time lapse images of effector T cell dynamics in the inflamed dermis.In this movie, the green adoptively transferred effector T cells can be observed moving throughout the dermal tissue. After the administration of antibeta one and antibeta three entogrin blocking antibodies through the catheter, however, the previously motile cells arrest within the dermis. The cells exhibit a decreased average velocity after antibody administration as well as a significant decrease in their meandering index.That is, the ration of the total displacement to the total track length. Autofluorescents from hair follicles, shadowing and autofluorescents from overlying hair, and air bubbles trapped between the ear surface and the cover slip, can all lead to imaging artifacts, which obscure the visualization of the T cells, and interfere with the automated image analysis software. In addition, using multiple sources of heat can cause tissue stability issues, leading to large oscillations during imaging that make interpreting the results difficult.Once mastered, imaging the CD4 T cells in the dermis can be completed in two to three hours, if the procedure has been performed correctly. While attempting this procedure, it's important to remember to follow the appropriate animal care and use guidelines for working with mice. This procedure can be adapted to visualize other T cell subsets or types of immune cells in the ear, using different models of infect, or different methods of inducing inflammation.After its development, intravital imaging paved the way for researchers in the field of immunology, to explore the dynamics of ongoing immune responses in living animals. Don't forget that working with a multi-phosphor laser can be extremely hazardous and that precautions, such as proper shielding, should always be taken while performing this procedure. After watching this video, you should have a good understanding of how to induce inflammation in a mouse ear dermis, how to use intravital imaging to capture the time lapse images of CD4 T cell activity before and after antibody administration.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Retrovirale Transductie van T-cel-receptoren in Mouse T-cellen

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

Concurrerende Homing Testen om de Gut-tropische T-cel-migratie Study

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Een kwantitatieve evaluatie van celmigratie door de Phagokinetic Track Motiliteit Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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

Real-time Live beeldvorming van T-cel signaleringscomplex Vorming

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

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritische cel-migratie met behulp van<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

Isolatie en Th17 Differentiatie van naïeve CD4 T-lymfocyten

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In vitro onderzoek naar de impact van immunoregulatoire Pathways op HIV-specifieke CD4 T-cellen Effectorfunctiemanipulatie Evalueer

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Transplantatie van Tail Skin aan allogene CD4 T-cel responsen bij muizen

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Muis Naïve CD4<sup&gt; +</sup&gt; T Cell Isolatie en<em&gt; In vitro</em&gt; Differentiatie in T cel subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In vitro differentiatie van humane CD4 + FOXP3 + geïnduceerde regulatoire T-cellen (iTregs) van naïeve CD4 + T cellen met behulp van een TGF-β-bevattende Protocol