This work was supported by funding received from the National Institutes of Health (1R01AI083269).

쥐의 연구는 노스 캐롤라이나 채플 힐 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인 된 모든 동물 보호는 IACUC 지침에 따라이었다. 신생아 및 성인 Thymi 1. 준비 <ol><li> 이전 기증자 쥐를 안락사에 대한 모든 시약 및 장비를 준비합니다. </li><li> 고압 증기 멸균 또는 기타 적절한 방법으로 수술 도구를 소독. 모든 수술은 멸균 조건을 유지하고, 오염을 방지하기 위해 층류 후드에서 수행되어야한다. 기증자 마우스에서 thymi의 추출을 위해 필요한 도구를 조립합니다. </li><li> 멸균 1X PBS (PH 7.4)와 60mm 요리를 입력하고 후드 내부의 얼음에 배치합니다. 이 전에 이식 기증자 마우스에서 절제 간단히 저장 thymi하는 데 사용됩니다. </li><li> 윤리 지침에 따라, 공동으로 자궁 경부 전위 다음 2 질식을 참수 성인 기증자 마우스로 신생아 기증자 쥐를 안락사. </li><li> R의 경우흉선의 emoval은 : 멸균 흡수 종이 타월에 지느러미 누운 위치에 마우스를 놓고 절개를하기 전에 70 % 에탄올로 스프레이. </li><li> 피부를 통해 중간 선 절개를하여 복부와 흉강을 노출. 흉강을 나타 내기 위해 가슴과 앞발을 통해 피부를 접​​습니다. </li><li> 우수한 종격동 노출과 흉강을 전방에 격막과 흉곽을 통해 두 가지 측면 절개를합니다. 흉선은 바로 위의 두 개의 흰색 로브로 볼 수 있고 마음에 인접한다. </li><li> 캡슐을 방해하지 않도록 특정하고, 미세 집게로 흉선 주위의 결합 조직을 떨어져 애타게. 집게와 흉곽을 다시 누른 상태에서 장기 아래 곡선 집게의 위치를​​ 수직으로 잡아 당겨 흉선의 두 로브를 추출 포셉의 두 번째 쌍을 사용합니다. 이는 신생아 마우스로부터 추출 thymi 해부 범위를 사용하여 수행 될 수있다. </li><li> 60mm 디에 흉선 배치SH 결합 지협을 통해 절단에 의해 얼음과 별도의 흉선 로브에 멸균 1X PBS (PH 7.4)를 포함. 캡슐이 손상되지 않도록 특정 만드는 흉선 로브에서 모든 파편을 제거하고 이식 섹션의 해당 번호로 흉선를 잘라. </li><li> 신생아 쥐에서 얻은 thymi를 조작하지 마십시오. 참고 :이받는 사람 마우스 (받는 사람 당 1 엽)의 최대 사용합니다. </li><li> 4-6받는 마우스의 최대로 성인 마우스로부터 수득 thymi 이식. 그림 1과 같이 신중하게, 성인 흉선 엽을 파악, 포셉 한 쌍을 사용하여, 수술 가위를 사용하여 세 개의 동일한 부분으로 흉선을 잘라. </li><li> 반복 각 기증자 마우스 1.3-1.4 단계를 반복합니다. thymi 노출 시간을 제한하기 위해, 한 번에 한 도너 흉선을 준비한다. </li></ol> 신장 캡슐에서 2 흉선 이식 <ol><li> 표 1에 예비 살균 장치를 조립한다. 적절한 무균기술은 오염 된 도구 나 시약에 이식받는 사람에게 노출되지 않도록하는 절차의 과정에서 활용되어야한다. </li><li> 이전 이식에, 무게 및 각받는 사람 마우스를 태그. </li><li> 층류 후드의 해부 현미경 및 마취 회로를 설정합니다. </li><li> 전기 면도기를 사용하여 수신자 마우스의 좌측 면도와 털이 절개하는데 사용 지역 주위에 남아 있도록. </li><li> 이소 플루오 란 기화기의 전원을 켜고 1 ~ 2 % 사이의 복용량을 사용하여 마우스를 마취. 이전 발가락 핀치 다음 반사의 부재를 확인하여 수술 절차를 시작하기에 적절한 마취를 확인. </li><li> 마우스가 제대로 마취 후, 건조를 방지하고, 다음과 같이 이식 마우스를 준비하고, 마우스 눈 연고 수의학 적용 </li><li> 면도면이 위를 향하도록 오른쪽 측면 누운 위치에 해부 현미경으로 마우스를 놓습니다. StartiNG 외과 영역의 중심에서, 포비돈 요오드 (betadine) 뒤에 전송 일회용 피펫 70 % 에탄올을 사용하여 원을 그리 분배. 절개를 만들기 위해 3 시간 전에 에탄올 / betadine 처리를 반복합니다. </li><li> 사용 해부 가위 직접 신장보다 1-2cm의 측면 절개를합니다. </li><li> 신장에 인접하여 결합 조직을 파악하고 근육 위에 놓 이도록 부드럽게 신장을 들어 올려 매체 집게를 사용. 근육을 통해 노출 된 신장과 장소에 유지하려면, 신장 장골을 방해하지 않도록 특별한주의를 기울이고, 신장 아래 매체 집게의 한 팔을 삽입합니다. </li><li> 조직의 탈수를 방지하기 위해, 신장과 신장 캡슐의 조작이 완료 될 때까지 각 단계 동안 멸균 1X PBS (PH 7.4)으로 신장 관개. </li><li> 미세 집게가 끼지과 신장에서 분리하는 부신에 가장 말단 신장의 가장자리 근처에 캡슐을 들어 올려 사용. </li><li> 18 게이지를 사용하여바늘은 기증자의 흉선 (그림 2A)의 준비 조각을 삽입하기에 충분히 큰 절개를합니다. 시간이 지남에 따라 그래프트의 전위를 방지하기 위해 가능한 한 작은 낭 절개를 유지 특정 확인. </li><li> 캡슐을 유지하는 것은 신장에서 멀리 당겨 동안, 캡슐 아래 이식을 삽입 가능 (그림 2B)로 피막 절개에서까지 앞으로 흉선을 밀어 미세 집게의 두 번째 쌍을 사용합니다. </li><li> 신장 아래에서 집게를 제거하고 부드럽게 절개를 통해 장소에 다시 신장을 반환합니다. </li><li> 복막 벽을 봉합 한 번 폐쇄 betadine을 적용하여 근육을 닫습니다. 봉합이 완료되면, 진피를 닫고 주변부 betadine을 적용 창상 클립을 적용한다. </li><li> 열 램프를 사용하여 예열 된 케이지에 마우스를 돌려줍니다. </li><li> 이 전체 의식과 이동성을 회복 할 때까지 각각의 수술 후 마우스를 모니터와 마우스 그쪽을 배치하지 마십시오(T)는 다른 동물과 케이지에 마취를 다음 회복하지 않았습니다. </li><li> 필요에 따라 수술 후 통증 관리의 약물과 쥐를 치료​​. 예컨대 1.6 mg / ml의 농도로 식수 아세트 아미노펜 등의 수술후 진통제와 마우스를 제공한다. </li><li> 반복 각받는 사람 마우스 2.3-2.9 단계를 반복합니다. </li><li> 1 시간 수술 후 내에서 정상적인 활동에 그 마우스 수익을 확인합니다. 이식 절차의 결과로서 발생하는 합병증을 결정 수술후 중량, 운동 능력뿐만 아니라, 각 마우스의 음주와 급전 활동을 모니터. 주 : 수술 전 중량뿐만 아니라, 이동도 변경 또는 먹는 음료 또는 합병증을 나타낼 수 실패에 비해 수술 후 체중 감소. </li><li> 꼬리 닉 제조업체의 사양에 따라 Lympholyte 세포 분리 미디어를 사용하여 림프구를 분리 한 다음을 통해 마우스를 출혈에 의해 말초 혈액에서 T 세포 재구성을 모니터링합니다. T 세포 characteriza 달성이러한 형광 활성화 숙신 이미 딜 에스테르 (8)과 같은 T 세포 마커 CD3, CD4, CD8 및뿐만 아니라 라이브 죽은 판별에 대한 특정 형광 복합 항체와 림프구를 염색하여 기. 도 3에 도시 된 바와 같이 분석 단봉, 생균 및 CD3 양성 세포상의 게이팅을 통해 달성 될 수있다. </li></ol>

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The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

T는 흉선 세포 발달에 중요한 이벤트가 1을 발생하는 기관이다. 상주 흉선 세포는 T 세포 수용체 (TCR) α 및 β 유전자의 재 배열시, 흉선 (2)의 피질 및 수질 지역 lymphostromal와 항원 제시 세포 (APC)와 일련의 상호 작용을 겪는다. 흉선 양성 선별은 호스트 주요 조직 적합성 복합체 (MHC) 클래스 I 및 II 분자 2-3의 컨텍스트에서 항원 성 펩타이드를 인식 흉선 세포를 생산하기 위해 피질 흉선 상피 세포 (TEC)에 의해 매개된다. 이후 흉선 음의 선택은 수질 TEC 또는 수지상 세포 (DC) MHC 클래스 I 및 II 분자 (3)에 의해 결합 된 자기 단백질로부터 유래 된 것을 본 펩타이드와의 상호 작용에 의해 구동 자기 반응성 흉선 세포의 정화를 수반한다. 이 프로세스의 최종 결과는 광범위한 스펙트럼 응답 할 수 성숙한 CD4 + 및 CD8 + T 세포의 풀의 설립이다외국 항원의 자기 단백질 4 최소한의 반응성을 전시한다. 흉선 선택 이벤트 효율 성숙 흉선, 골수 및 피질 TEC, 흉선 DC의 서브셋 조성물의 주파수 및 흉선 전구체 (3)의 소스를 포함 요인의 호스트에 의해 영향을 받는다. 특히, 비정상적인 흉선 선택은 각각 장애인 부정적 또는 긍정적 인 선택에서 발생하는자가 면역 5 또는 면역 결핍 병리,이 발생할 수 있습니다. 흉선 선택을 조절하는 분자 이벤트는, 그러나, 제대로 이해된다. 체외은 재 응집 흉선 기관 배양 (RTOC) 6으로 접근, 흉선 선택과 관련된 기본 이벤트를 분석하는 데 유용 할 수 있지만 완전히 진행의 역학을 요점을 되풀이하지 입증했다 생체 이벤트에서. 따라서이 흉선 이식 기반 접근법은 생체 (7)에 더 연구 흉선 세포 선택 이벤트에 설립되었다 </s&gt;까지. 이 프로토콜은 면역 결핍 SCID받는 마우스로 신생아와 성인 기증자 마우스에서 thymi 이식에 대해 설명합니다. 이 기술은 양극과 음극 흉선 선택뿐만 아니라, 개체 발생 동안 다양한 T 세포 서브 세트의 흉선 출력을 조절 메커니즘의 연구를 허용한다. 최근에,이 접근법은 흉선 선택의 효율성은 초기 자기 반응성 T 세포의 증가 된 발달 선도 마우스에서 생후 제한된다는 것을 입증하기 위해 사용하고, 외부 항원 7 감소 T 세포 레퍼토리 특정되었다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Decon Laboratories</td>
			<td>2705HC</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>pH 7.2</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (1.0 metric)</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>18&#34; (45cm)</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips (9mm)</td>
			<td>Clay Adams&#174; Brand</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr>
			<td>Mouse anti-CD3 Ab</td>
			<td>eBioscience</td>
			<td>11-0031-85</td>
			<td>Clone: 1452C11</td>
		</tr>
		<tr>
			<td>Mouse anti-CD4 Ab</td>
			<td>eBioscience</td>
			<td>48-0042-82</td>
			<td>Clone: RM4-5</td>
		</tr>
		<tr>
			<td>Mouse anti-CD8 Ab</td>
			<td>eBioscience</td>
			<td>25-0081-82</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Lancet</td>
			<td>Medipoint</td>
			<td>Goldenrod 4mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td>
			<td>Invitrogen</td>
			<td>P30253</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round botton polypropylene plates</td>
			<td>Corning</td>
			<td>3365</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2mL polypropylene cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tubes</td>
			<td>BD</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>40uM Nylon Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution, EM Grade</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Puralube Optical Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0138063</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td>60mm x 15mm, Sterile</td>
		</tr>
	</tbody>
</table>

isolation and transplantation of different aged murine thymic grafts.

흉선 선택의 효과를 조절하는 메커니즘은 잘못 정의 된 남아 있습니다. 생체가 자신과 외부 항원에 특이적인 T 세포의 발달 및 선택 분석 여기에 제시된 방법이 있습니다. 방법은 면역 결핍 SCID받는 사람에 다양한 세 생쥐에서 파생 된 흉선 이식의 이식을 수반한다. 비교적 짧은 시간 동안받는 완전히 주입 흉선 이식편 유래의 T 세포로 재구성된다. 흉선 세포 만 분리시 흉선 시드가 선택을 거쳐 성숙한 T 세포로 발달. 이와 같이, 연령 의존적 흉선 이벤트 함수로서 접목 T 세포의 특성 및 특이성의 변화가 평가 될 수있다. 전문 기술이 성공적 흉선 이식 필요하지만,이 방법은 생체 내 비정상적 흉선 기능 및 / 또는 homeostas의 결과로 인한 또는 병리 광범위한 연구하는 독특한 전략을 제공한다이다.

이 절차의 성공은 최소의 외상 수술뿐만 아니라, 신장 캡슐 밑에 그래프트의 정확한 위치에 의존한다. 도 1에 도시 된 바와 같이 흉선 그래프트 적절히 확보 후속 이식 흉선 섹션 크기로 절단되어야한다.도 1의 개략도에 따라, 신생아 나 성체 생쥐 thymi 일관되고 재현 T 세포의 생착 결과 성공적인 피막 하 이식에 사용될 수있다. 언급 한 바와 같이, 신장 캡슐 밑에 그래프트...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

분리 및 다른 세 쥐 흉선 이식의 이식.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Research

JoVE Journal

Immunology and Infection

13.7K 조회수.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

비디오: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

연구

면역학 및 감염병학

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

쥐과 잇몸 세포의 분리, 처리 및 분석

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

쥐과 림프절 기질 세포의 분리

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

쥐 소장 림프구의 분리 및 유세포 특성

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

준비 및의 Organotypic 흉선 슬라이스 문화의 응용 프로그램

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

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B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

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Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...