저자는 라이브 영상에 대한 도움말은 로체스터 다중 광자 현미경의 핵심 시설의 대학 감사합니다. DJF에 NIH AI072690 및 AI02851에서 지원; MGO에 AG 및 AI089079에 AI114036.

저자는 공개 아무것도 없어.

의미 여기에서 우리는 그대로 마우스 귀 진피에 전달, 항원 특이 이펙터 Th1 세포의 4D 시각화를위한 완벽한 프로토콜을 제시한다. 이 방법은 여러 가지 이유로 몇몇 현재 영상 방식에 비해 많은 장점을 제공한다. 복부 귀 진피 촬상함으로써 다른 피부 부위를 포함하는 촬상 프로토콜이 필요 제모를 포기 할 수있다. 제모 일반적으로 온화하지만, 이들은 피부 장벽 (42)에 대한 면역 반응을 자극 할 수 43,44 프로세스를 중단을 야기하는 것으로 나타났다. 또한 진피 또는 피하을 노출 침습 수술 절차를 피함으로써,이 프로토콜은 손상에 의한 염증과 진피로 호중구 (37) 및 다른 면역 세포의 빠른 채용을 방지 할 수 있습니다. 키 분자에 대하여 차단하는 항체를 제공하기이 시스템에 정맥 카테터의 사용은 다이나 실시간 심문 허용CD4 T 세포의 마이크 동작. 이 촬상 프로토콜의 사용은 시험 관내 시스템 (8)에 검출되지 않은 CD4 T 세포 간질 운동성 30 중요 요건을 밝혔다. 절차에서 중요한 단계 어떤 이미징 프로토콜에 필수적인 단계는 이미징을위한 안정적인 조직 준비를 보장한다. 이 기포가 없는지 귀 및 커버 슬립 사이에 충분한 PBS가있는 것이 중요하고 귀 테이프가 유리 탈 부착되는 원인로서 유리와 접촉하지만, 그렇게하지. 마찬가지로, 플랫폼으로 커버 슬립과 시간이 지남에 풀어에서 테이프를 방지 할 수있는 진공 그리스를 들고 테이프 사이의 접촉을 피하는. 온도는 진동을 방지하고 열 수축 또는 플랫폼 재료의 확장에서 표류 일정하게 유지해야합니다. 제한 사항 및 수정 45 시간 경과 이미징 모델로 사용되고있다 기인 것이 바람직하다. 그러나, 귀 상주 면역 인구는 측면 또는 풋 패드 (46)에 피부를 구별하고, 마우스 귀는 다른 사이트의 47에 비해 뚜렷한 혈관의 속성이 있습니다. 따라서, 몇몇 응용을 위해, 다른 피부 부위에 이어 촬상 데이터의 비교가 어려울 수있다. 이 프로토콜은 또한 혈류에서 진피 내로 이송 이펙터 T 세포의 효과적인 이동을 필요로한다. 이것은 그들이 격자 공간에 들어갈 수 없으므로 유도 또는 혈관 외 유출에 결함이있는 셀을 사용하는 능력을 제한한다. 넘쳐 흐름이것은 생체 내에서 혈관 외 유출 겪는 세포들의 위치 파악 또는 동작 요점을 되풀이되지 않을 이러한 방식으로 일부 기계적 손상과 세포의 전달을 발생할 수 있지만, 직접 이어 37,48 진피로 세포를 주입하여 무시 될 수있다. 또한 여기에 또는 노 C57BL / 6 티르 C-2J 마우스 사용 된 BALB / c를 변형 등의 영상 실험에 대한 비 착색받는 사람 마우스 사용을 고려하는 것이 중요합니다. 착색 된 마우스에서 멜라닌은 매우 autofluorescent 것에 더하여, 광자, 레이저 (37)로부터 심지어 비교적 저전력 여기하에 가열한다. 이 결과를 복잡하게 피부와 이후의 염증 (36) 또는 형광 반점 (31)에 열 손상을 일으킬 수 있습니다. 이는 다중 파라미터 촬상 실험에 사용할 수있는 형광체를 제한 할 수있다. 그러나 매우 밝거나 매우 표현 형광 분자가 모두 낮은 레이저 파워에 효율적으로 흥분 될 수 있습니다착색 된 생쥐에서 효과적인 시각화 때문에. 미래의 응용 프로그램 이것은 비 침습성 과정이기 때문에, 용이하게 연장 기간에 걸쳐 순차적으로 촬상 쥐 종 연구에 적용 할 수있다. 기간 동안 큰 3 ~ 4 일이 사라질 것입니다 여기에 설명 된대로 형광 표시하지만, 내생 형광 세포 또는 형광 기자 세포의 사용은이 문제를 제거합니다. 사실, 우리는 이전에 30,49 예티와 IL-4 기자가 50 4get IFNγ 기자를 포함하는 사이토 카인 기자, 베어링 vivo- 생성 된 항원 특이 이펙터를에 추적이 프로토콜을 사용하고 있습니다. 우리는 또한 CD4-Cre 호텔 ROSA26 스톱 floxed EYFP 형광 기자 마우스 (30)에 내생 CD4 세포를 시각화했다. EYFP 다른 형광 단백질의 형광 특성 등 CFSE 화학 염색에서 다르기 때문에, 촬상 파라미터에 대한 수정이 필요할 수있다효율적인 시각화를위한. 이러한 일부 희미 형광체의 형광 신호를 향상시킬 수있는 화소 체류 시간을 증가하고, 관찰 될 수있는 임의의 광표백을 완화 할 수 시간 경과 이미지의 길이를 감소하는 등의 변화. 900 nm의 여기에서, 우리는 이전에 짧은 촬영 간격을 통해 CFSE, CMTMR, 또는 EYFP의 중요한 광표백을 관찰하지 않았습니다. 이 절차는 CD4 이펙터 T 세포 운동성의 역학을 측정에 집중하지만, 본 응용에 한정되지 않는다. 작업 항원은 세포 또는 51,52 촬상 전에 조직 구조 라벨 진피 내로 형광 리포터 생쥐 형광 복합 항체 주사를 사용하여 세포를 제시하여 이펙터 T 세포의 동적 상호 작용을 측정하기 위해 현재 진행 중이다. 이 프로토콜은 차단 항체를 제공하기 위해 카테터를 사용하는 경우이지만, 또한 반면 이미징, 소분자 저해제를 포함하는 다른 화합물은 수투여 될 수있다. 궁극적으로,이 프로토콜은 비 침습적 방법으로 생체 내에서 시간이 지남에 따라 면역 동성을 측정하기 위해가요 성 플랫폼을 제공한다.

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

이펙터 기능을 수행하는 CD4 T 세포의 능력은 같은 아직 정의되지 않은 메카니즘을 통해 염증 말초 조직에서 이들 세포를 신속하고 효율적인 이동에 의존한다. 면역계 연구에 다 광자 현미경의 적용은 그대로 조직 내 면역 반응의 동역학을 측정하는 도구를 제공한다. 여기에서 우리는 염증 마우스 귀 진피에 CD4 T 세포의 비 침습적 생체 내에 광자 이미징을위한 프로토콜을 제시한다. 맞춤 이미징 플랫폼을 이용하여 정맥 카테터 운동성에 관여하는 중요한 분자 성분에 대한 항체를 차단의 추가를 통해 실시간으로 이러한 세포를 심문하는 기능, 진피 간질에서 CD4 T 세포 역학 시각화 가능하다. 이 시스템은 체외 모델과 외과 적 침습 이미징 절차 둘다 장점을 제공한다. 운동에 대한 CD4 T 세포에 의해 사용되는 경로를 이해하는 것은 궁극적으로 BASI에 대한 통찰력을 제공 할 수있다C의 CD4 T 세포의 기능뿐만 아니라 만성 감염에서 양자 면역 질환의 발병 기전 및 병리.

면역 환경 변화없이 제자리에서 면역 반응을 연구 할 수있는 능력은 염증 조직으로 T 이펙터 세포의 실시간 상호 작용을 연구하는데 필수적이다. 도 1A 및 B에 요약 된이 프로토콜에 의해 그대로 이어 진피 이미징, 진피 간질 양도 형광 표지 T 이펙터 세포의 시각화를 허용한다. 이것은 고해상도 (도 1C) 및 저속 (도 1D, 영화 1) 염증 진피 이펙터 T 세포 역학 이미지 모두를 허용한다. <img alt="그림 1" src="/files/ftp_upload/53585/53585fig1.jpg" /> 도 1 고해상도 및 손상 피부 간질에서 이펙터 T 세포 4D 촬상. (A) 실험 개요. 귀 위해 준비 할의 (B) 사진영상 (레드 라인)의 에멀젼의 사이트 (검은 점선) 및 최적의 공간 이미징을위한 에드는 지적했다. 섬유 성 콜라겐 (청색) 및 텍사스 레드 덱스 트란으로부터 고해상도 CFSE 표지 된 Th1 세포 (녹색)를 보여주는 스택 번째 고조파 신호 (C) 최대한 차원 투영 혈관 (적색) 표지. 흰색 화살표는 여러 autofluorescent 모낭 중 하나를 나타냅니다. 스케일 바는 대표 50 μm의 (D)에 CFA-염증이 진피에서 30 분 동안 추적 Th1 세포의 철새 경로. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 이 촬상 프로토콜 촬상 동안 흡입 마취의 전달을위한 하우스 구축 된 특수 촬상 플랫폼의 사용뿐만 아니라 적응 노즈콘을 필요로한다. 이미징 플랫폼 (그림 2A-B)는 O를로 구성상승 된 중앙 섹션 알루미늄베이스 플레이트 F. 이 제기 부분은 아크릴 늘어서 삽입 부분이 압축 잠재적 얇은 조직에 손상을주지 않고 귀에 지원을 제공하는 느낌이있다. 우리의 설치의 형상은 촬영하는 동안 흡입 마취를 제공하는 유연하고 조정 원추형 두부의 건설이 필요합니다. 플렉시블 호스 (도 2c)의 50mm 부분에 연결된 변성 microcentrifuge 관으로 이루어진이 원추형 두부는 개질 마이크로 원심 튜브로 이루어지는 홀더 (도 2d)를 제 위치에 고정 후크 및 루프 파스너를 통해 이미징 플랫폼에 부착 된 . 후크 및 루프 패스너의 사용은, 마우스의 두 귀를 이미지화 할 수 있도록 원추형 두부 조립체의 재배치를 허용하고, 최적 개별 마우스에 대해 위치 될 수있다. <img alt="그림 2" src="/files/ftp_upload/53585/53585fig2.jpg" /> 그림 2. 동등생체 내에 이미징의 pment. 맨 (A) 및 측면 (B)보기에 맞춤 제작 된 영상 플랫폼입니다. 이소 플루 란 관리 (C)과 원추형 두부 홀더 (D)에 대한 원추형 두부. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 꼬리 정맥 카테터는 혈관에 라벨을 높은 분자량 덱스 트란과 같은 항체 및 혈관 밖으로 확산 할 수있는 다른 작은 분자, 또는 더 큰 형광 분자를 관리하는 순환에 지속적으로 액세스 할 수 있습니다. 안티 - β 1 항 β 카테터를 통해 항체를 차단 3 인테그린, 진피 내에서 이전에 운동성 세포의 체포 (동영상 2)의 100 μg의 투여 후. 이러한 세포는 항체 후 감소 된 평균 속도를 가지고<html> 투여 (도 3a)뿐만 아니라, 지그재그 지수의 상당한 감소, 총 트랙 길이 (도 3b)의 전체 용량의 비율. <img alt="그림 3" src="/files/ftp_upload/53585/53585fig3.jpg" /> 안티 - β 1 항 β 3 항체의 그림 3. 관리는 CFA-염증이 피부 Th1 세포의 이동을 억제한다. (A) Th1 세포의 평균 속도를 이전과 100 μg의 안티 - β 1 항 β 3 인테그린의 투여 후 항체를 차단. Th1 세포 전에 항체 봉쇄 이후의 (B) 사행 인덱스입니다. 약 100 추적 세포는 전에 하나의 대표 실험에서 한 번의 마우스에서 항체 봉쇄 한 후 이미지에서입니다. 맨 휘트니에 의해 통계.에스 / ftp_upload / 53585 / 53585fig3large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. 모발 귀의 표면으로부터 제거하지 않기 때문에 모발의 화상 아티팩트는 공통이다. 들은 T 세포를 모호 자동화 된 이미지 분석 소프트웨어를 방해 할 수있는 모낭 (도 4a) 및 상부 머리 (도 4b)에서 음영 및 형광도는 형광도로부터 가능한 한 피해야한다. 마찬가지로, 귀 표면 및 커버 슬립 사이에 갇힌 기포 영상 아티팩트 (도 4C)으로 이어질 수있다. 귀 적절한 제제는 임의의 잔류 기포의 개수와 크기를 최소화한다. <img alt="그림 4" src="/files/ftp_upload/53585/53585fig4.jpg" /> 그림 4. 헤어 형광도 가난한 귀 사전에서 일반 유물paration. (A) Autofluorescent의 모낭. (B) Autofluorescent 머리와 머리 여포 (녹색) 및 이미지의 어두운 라인의 원인이 상부 머리 그림자, 콜라겐 (흰색). 변위, 표피 각화 autofluorescent 가장자리 (점선)을 도시 기포로부터 (C) 아티펙트. 스케일 바는 50 μm의를 나타냅니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 또한, 열 다중 소스의 사용은 열팽창 및 수축에 대한 조직의 안정성에 문제가 발생할 수있다. 잘못된 온도 설정은, 예를 들어, 어려운 결과 (동영상 3)의 해석을 할 수 이미징 동안 큰 진동으로 이어질 수 있습니다. 일정한 온도를 제공하고, 안정성을 최대화하는 시스템에 대한 최적의 설정을 결정하는 것이 필수적이다. Ultimat엘리는 온도 조절 이미징 챔버는 온도 변화에서 변동성을 제거하는 가장 좋은 방법입니다. <img alt="영화 1" src="/files/ftp_upload/53585/53585movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie1.mov" target="_blank">CFA-염증이 진피에 마이그레이션 영화 1. Th1 세포는 (오른쪽 다운로드 클릭).</a> 30 분의 시간 경과 이미지가 피부 콜라겐 네트워크에서 마이그레이션하는 Th1 세포 (녹색) (두 번째 고조파 발생, 파란색)을 게재합니다. <img alt="영화 2" src="/files/ftp_upload/53585/53585movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie2.mov" target="_blank">β 1의 봉쇄와 3 인테그린을 β에 따라 영화 2. Th1 세포의 체포 (오른쪽 다운로드 클릭).</a> 세포 (녹색)는 administ 전에 30 분 동안 몇 군데 있었다차단 항체를 정량 한 다음, 동일한 위치에서 또 다른 20 분 동안 묘화. <img alt="영화 3" src="/files/ftp_upload/53585/53585movie3.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie3.mov" target="_blank">가난하게 제어 가열 플레이트 3. 진동 영화 (오른쪽 다운로드 클릭).</a> Th1 세포 (녹색) 및 두 번째 고조파 세대 (파란색)의 30 분의 시간 경과 이미지를. 이 이미지를 수집 한 후, 사용한 가열판은 이미징 플랫폼과 후속 열 팽창 및 수축의 온도에서 진동을 일으키는 결함 서모 스탯을 보유하고 있음이 발견되었다.

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

염증이 진피의 이미징 CD4 T 세포 간질 마이그레이션

염증 부위에서 이펙터 CD4 T 세포의 삽입 운동을 제어하는​​ 메카니즘은 상대적으로 알려져 있지 않다. 우리는 시각화 현장에서 이러한 세포의 동적 거동의 연구를 허용 염증 귀 진피의 시험 관내 -primed CD4 T 세포에서 조작하는 비 침습적 방법을 제시한다.

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 ...

imaging-cd4-t-cell-interstitial-migration-in-the-inflamed-dermis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.6K 조회수.  University of Rochester School of Medicine and Dentistry, Rochester, NY.  염증 부위에서 이펙터 CD4 T 세포의 삽입 운동을 제어하는​​ 메카니즘은 상대적으로 알려져 있지 않다. 우리는 시각화 현장에서 이러한 세포의 동적 거동의 연구를 허용 염증 귀 진피의 시험 관내 -primed CD4 T 세포에서 조작하는 비 침습적 방법을 제시한다. 

비디오: Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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

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 presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

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 ...

연구

면역학 및 감염병학

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

굿 - 트로픽 T 세포의 마이 그 레이션을 공부 경쟁력 호밍 Assays

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

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokinetic 트랙 운동성 분석에 의한 세포 마이그레이션의 수량 평가

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

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

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

T 세포의 실시간 라이브 영상은 복잡한 형성을 신호

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

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

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

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

사용 모니터링 수지상 세포 이동<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H 자기 공명 영상

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

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 including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

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

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

분리 및 나이브 CD4 T 림프구의 TH17 분화

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

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 upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

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

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

시험 관내 분석에서 HIV 특정 CD4 T 세포의 이펙터 기능에 면역 경로의 영향을 평가하기

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

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

마우스에 동종 CD4 T 세포 반응을 연구하기 위해 테일 피부 이식

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

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.

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

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

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell 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 of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

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

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

마우스 나이브 CD4<sup&gt; +</sup&gt; T 세포의 분리 및<em&gt; 시험관</em&gt; T 세포 하위 집합으로 차별화

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