저자는 도움이 토론을 위해 컬럼비아 대학에서 미생물학과 면역학의학과에서 박사 보리스 Reizis 감사하고 싶습니다. 우리는 또한 유동세포계측법 및 비디오 편집과 지원 루 후앙과 기술 지원에 대한 동물용 의약품의 대학, 코넬 대학에서 미생물학과 면역학의학과에서 박사 마가렛 바이노 박사 로드먼 겟첼 및 Deequa Mahamed 감사하고 싶습니다. / - - 생쥐 저자는 또한 p53의를 사육 및 genotyping에 대한 와이즈 실험실에서 자신의 중요한 원고와 비디오 제작의 검토, 그리고 스테파니 Yazinski에 대한 듀하멜 와이즈 실험실의 구성원을 감사하고 싶습니다. 동물 실험은 코넬 대학 기관 애니멀 케어 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다. 이 연구는 농업의 미국학과, 협동 상태 연구, 교육 및 연장 서비스, 동물 건강 및 질병 연구 프로그램 (검정 고시 및 RSW)을하고 NIH 보조금 R03 HD058220 및 R01CA108773 (RSW)을 제공한 자금에 의해 부분적으로 지원되었다. 비디오 듀하멜 &amp; 와이즈 연구소의 인력에 의해 자체 시설을 사용하여 제작되었습니다.

1. 종양의 해부 <ol><li> 종양 절개 들어, 깨끗한 종이 타월이나 일회용 외과 드레이프는 해부 패드에 위치하고 있으며 70 %의 에탄올로 뿌렸어요. </li><li> 이 프로토콜에 대한 생쥐가 일상적으로 CO 2 euthanized 있습니다. 절개 패드에 euthanized 마우스를 놓고 에탄올과 동물의 양쪽을 스프레이. 마우스는 다리를 통해 삽입 70 % 에탄올로 철저하게 뿌려놓은 4 개 이상의 푸시 핀을 사용하여 절개 패드에 장소에서 개최됩니다. </li><li> 정중선을 통해 피부는 기본 근육을 피하와 본문 충치에 관통하면서, 멸균 가위를 사용하여 치골에서 머리의 기본으로 들어가있다. </li><li> 무균 가위와 내부 장기를 손상하지 않도록주의하는 포셉의 새로운 쌍을로 복강을 엽니다. </li><li> 해부 및 손상 혈관없이 종양을 나타내기 위해 갈빗대을 왜곡하여 흉강을 엽니다. </li><li> 다른 장기에서 종양을 분리하고, 차가운 PBS로 작품 전체 종양의 약 40~50%를 (약 최대 1.5 X 0.8 X 0.8 cm) 전송 얼음이 있었죠. </li><li> 종양의 나머지 부분은 다른 assays 저장 수 있으며 완벽한 검시이 시간에 수행할 수 있습니다. </li></ol> 2. Culturing의 림프종 세포 <ol><li> 70 % 에탄올과 biosafety 캐비닛에 전송으로 해부하는 종양을 포함하고있는 튜브를 스프레이. </li><li> 멸균 150mm 2 페트리 접시에 배지 10 ML을 추가합니다. </li><li> 피펫과 부드러운 흡입하여 페트리 접시에 종양의 조각을 전송합니다. </li><li> beveled 측면을 마주보고 두 멸균 18 게이지 바늘을 구부. </li><li> 벤트 바늘을 사용하여 종양을 떼어 놓다. </li><li> 소용돌이 접시를 부드럽게 탭 및 종양의 큰 조각을 피하면서, 측면에 세포 현탁액을 수집하는 각도로 놓으십시오. </li><li> 종양의 큰 조각을 피하면서 50 ML 원뿔 튜브에 세포 현탁액과 전송 대기음. </li><li> 종양의 큰 조각을 피하는 동안 같은 튜브에 매체 및 전송의 10 ML과 접시를 씻으십시오. </li><li> 부드럽게 lymphocytes와 stromal 세포로 구성된 세포 현탁액을 혼합하고 T - 75 조직 배양 플라스크에 T - 25 조직 문화 플라스크 10 ML에 5 ML을 전송하기만하면됩니다. 부드럽게 소용돌이 flasks합니다. </li><li> 2.5 ML 최종 볼륨 (문화 선박의 여러 유형을 사용하여 서로 다른 밀도에 culturing 세포 T 세포 라인을 확립 가능성을 극대화) 2, 4, 10100 1000 배 dilutions를 만들기 위해 나머지 세포 현탁액을 사용합니다. </li><li> 6 잘 접시의 각각의 우물에 각 희석의 플레이트 2.0 ML. 부드럽게 소용돌이 접시합니다. </li><li> 공기 humidified CO 2 분위기에서 48 H에 대한 세포를 품어. </li></ol> 3. 림프종 세포를주고 <ol><li> 부화 48 H 후, 세포의 최소한의 방해와 벽 함께 T - 75 플라스크에 T - 25 플라스크와 매체의 10 ML 중간의 5 ML를 추가합니다. </li><li> 세포의 최소한의 방해와 벽을 따라 6 잘 플레이트의 각 물론 중간 2 ML을 추가합니다. </li><li> 추가 72 H.에 대한 세포를 품어 </li></ol> 4. 림프종 세포를 Passaging <ol><li> 초기 통로 <ol><li> 원래 flasks 및 접시 (첫 번째 구절 중에 약 3x106 / ML의 농도에서 세포 밀도를 유지의 성공적인 구축을 촉진 유지하면서 부화의 다섯 번째 하루의 끝에서, 세포는 1:1 비율로 passaged 아르 연속 세포주). <ol class="lalpha"><li> T - 25 플라스크의 경우, 매체의 5 ML을 포함하는 새로운 플라스크에 세포 현탁액의 5 ML을 전송. 원래 플라스크에 매체의 5 ML을 추가합니다. </li><li> T - 75 플라스크 들어, 중간의 10 ML을 포함하는 새로운 플라스크에 세포 현탁액의 10 ML을 전송. 원래 플라스크에 배지 10 ML을 추가합니다. </li><li> 6 - 잘 접시, 각 우물에 중간 2 ML을 포함하는 새 접시의 해당 우물에 각 우물에서 세포 현탁액 2 ML을 전송. 원래 우물의 각 우물에 미디어 2 ML을 추가합니다. </li></ol></li><li> 추가 72 H. 모든 flasks과 접시를 품어 우리의 경험에서 대부분의 세포 라인은 이러한 통로 한 문화 (첨부된 stromal 세포를 통해 느슨하게 자기편 서스펜션 셀 집계의 존재가 설립 세포주의 초기 표시이다)에서 설정됩니다. </li><li> 적게 또는 전혀 느슨하게 자기편 서스펜션 세포 집계와 문화, 신중하게 중간마다 72 H.의 약 50 %를 대체하여 flasks이나 접시를 피드 원래 문화의 세포를 제거 방지하기 위해, 문화 선박이 뜨는 매체의 부드러운 열망 전에 중력에 의해 아래에있는 세포의 퇴적물을하도록 서있는 최소한 5 분 그대로 남아 있습니다. </li></ol></li><li> 설립 세포 라인을 유지 <ol><li> 설립 셀 라인은 새로운 flasks 및 플레이트로 passaged 있습니다. T - 25 T - 75 flasks 들어, 원본 또는 5 ML을 추가중간 5 ML에 통과 한 현탁액. 6 - 잘 접시 들어, T - 25 플라스크에 중간 7 ML에 원본 또는 통로 한 셀 서스펜션 3 ML를 추가합니다. 피드 신선한 배지로 제거 볼륨을 대체하여 원본 또는 통로 한 문화를 유지합니다. </li><li> 통로는 새로운 T - 25 flasks로 셀 라인을 설립하였습니다. 셀 라인은 18-24 H.의 두 배로 시간을 문화가 일상적으로 1 3x106 / ML의 밀도 유지, 3 일마다 passaged 있습니다. 10 ML까지 할 수있는 새로운 매체 이전에 통과 문화의 필요한 볼륨을 추가합니다. 피드와 원래의 문화를 유지합니다. </li><li> 몇 구절 후, 비 점착 성의 세포가 자기 점착 성의 stromal 세포없이 유지되며, 자기편 세포의 작은 숫자는 각 통로로 이월됩니다. 이 시점 이외에도, 문화가 T - 25 flasks을 처리하지 않는 조직 문화를 유지하실 수 있습니다. </li></ol></li></ol> 5. 림프종 세포 라인을 동결 복구 <ol><li> 셀 라인 20% FBS 및 10 % DMSO를 포함하는 신선한 RPMI 1640 배지에 냉동 수 있습니다. </li><li> T - 25 플라스크 (약 1.5x10 6 셀 / ML)에 48 H 옛 문화의 세포는 상온에서 5 분 1,500 rpm으로 centrifuged 있습니다. 폐기 뜨는 후, 세포는 추위 동결 중간 2 ML에 다시 일시 중지됩니다. 각각의 플라스크에서 세포 현탁액의 두 1.0 ML 볼륨 즉시 -80시 냉동, 동결 튜브에 배치됩니다 ° C, 그리고 그 다음날 액체 질소로 전학 왔어. </li><li> 전지는 빠른 해동하고, 신선한 문화 매체 10 ML에있는 내용을 resuspending하여 액체 질소 저장에서 복구됩니다. 또는, 갓 해동 세포는 신선한 문화 매체의 10 ML에 resuspending 전에 DMSO를 제거하는 따뜻한 문화 매체에 한 번 세탁하고 있습니다. 세포는 이후 통과하기 전에 최소한 72 H에 대한 양식입니다. </li></ol> 6. 대표 결과 ATM은 - / - T - 세포 라인이 프로토콜을 사용하여 개발된 및 지정 DWJ - ATM - 1 반 인 경우에는 3 번 CD - FITC, 안티 - CD4 - APC 및 안티 CD8 - PE 및 유동세포계측법 분석 (Figure1)와 얼룩에 의해 특징되었습니다. T - 세포의 90 % 이상 인 경우에는 3 번 CD, CD4 및 CD8 트리플 긍정했다. <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="그림 1" /> 그림 1.이 프로토콜을 사용하여 개발된 T - 세포 라인의 특성화. / - - 설립 ATM의 세포 표면 마커 세포 라인 DWJ - ATM - 1과 얼룩을 특징으로했다 지정한 안티 인 경우에는 3 번 CD - FITC, 안티 - CD4 - APC, 안티 - CD8 - PE 및 유동세포계측법 분석. 간단히, 1X10 6 신선한 세포는 차가운 PBS (1500 RPM / 5 분)에 한 번 씻어, 그리고 4 incubated 있었다 ° 1 % BSA와 PBS에서 1:200 희석에서 항체 칵테일 (각 항체의 μL 200 10 분 C. 신선한 이상 분석을 위해 고정. 수정된 세포가 4 % (권 / 권) 최종 농도에 추가 중립 버퍼 포르말린에 의해 준비 및 원심 분리에 의해 수확 30 분 (4 ° C에서 incubated하는 동안 세포는 즉시 검사 수있는 1,500 RPM / 5 분) 및 1 % BSA와 PBS 200 μL에 resuspended. 1 X 10 4 세포의 총 BD 외과 Calibur 분석기 및 CellQuest 소프트웨어 (BD ​​Biosciences, 산호세, CA)를 사용하여 분석되었다.

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관심 없음 충돌 선언하지 않습니다.

이 프로토콜에서는, 우리는 두 개의 다른 마우스 genotypes에서 murine T - 세포 라인을 확립하기위한 자세한 절차를 제공한다 - / - [FVB/N- ATM tm1Led / J]와 p53의 - / - ATM [129/S6- Trp53 tm1Tyj / J] . / - - 세 (5 시도 아웃) p53의 - / -이 프로토콜, 6 총 (7 시도 아웃) ATM을 사용하여 murine T - 세포 라인은 우리 연구실에서 설...

<ul><li> 아이스 버켓 </li><li> 70 % 에탄올 </li><li> 알루미늄 호일과 핀이으로 덮여 거품 블록으로 구성된 해부 패드 </li><li> 작은 무균 가위와 집게 두 세트 </li><li> 무균 150mm 2 페트리 접시 (써모 피셔 과학, 월섬, MA;. 고양이 # 08-757-13) </li><li> 멸균 18 게이지 바늘 </li><li> 50 ML 원뿔 튜브 (코닝 주식 회사, 코닝 NY, 고양이 # 430829) </li><li> 10 % 포르말린 버퍼 </li><li> 조직 문화 flasks <ul><li> T - 25 (TPP, Trasadingen, 스위스, 고양이 # 90025) </li><li> T - 75 (TPP, Trasadingen, 스위스, 고양이 # 90075) </li></ul></li><li> 6 - 잘 접시 (코닝 주식 회사, 코닝 NY, 고양이 # 3506) </li><li> 멸균 인산은 생리 산도 7.2 (PBS)를 버퍼 </li><li> 문화 매체 <ul><li> 25 MM HEPES, L - 글루타민은 (Lonza, 워커스빌, MD, 고양이 # 12 - 115F) 200 MM와 RPMI 1640 다음과 보충 : <ul><li> inactivated 10 % 열 (56 ° C, 30 분) 태아 소 혈청 (FBS; Hyclone, 로건, UT, 고양이 # SH30088.03) </li><li> 1퍼센트 페니실린과 스트렙토 마이신 (Mediatech, 머내서스, VA, 고양이 # 30-002 - CI) </li><li> 1퍼센트 불필요한 아미노산 (Mediatech, 고양이 # 25-025 - CI) </li><li> 55 MM 2β - 메르 캅 토 에탄올 (시그마, 세인트 루이스, MO, 고양이 # M752) </li></ul></li></ul></li><li> 디메틸 sulfoxide (DMSO, Calbiochem, 라 호야, CA, 고양이 # 317275) </li><li> 항체 (eBioScience, 샌디에고, CA) <ul><li> 안티 인 경우에는 3 번 CD - FITC (cat. # 11-0031-85) </li><li> 안티 - CD4 - APC (cat. # 17-0041-83) </li><li> 안티 - CD8 - PE (cat. # 12-081-85) </li></ul></li><li> 보빈 알부민 혈청 (BSA, 생명 기술, 그랜드 아일랜드, NY,. 고양이 # 11018-017) </li><li> Concanavalin A (시그마 고양이 # C5275) </li><li> 1.8 ML 동결 튜브 (Nunc, 로스킬레, 덴마크, 고양이 # 368632) </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

설립 세포 라인은 연구 실험 동물의 사용을 줄일 수있는 중요한 연구 도구입니다. 특정 같은 ATM과 같은 유전자 변형 생쥐의 변종 - / -와 p53의 - /이 - 일관되게 일찍 삶 1,2에서 thymic 림프종을 개발하고, 따라서, murine T - 세포 라인의 유도를위한 신뢰할 수있는 소스로 검색할 수 있습니다. 여기는 이전의 프로토콜 1,3에서 설명한 interleukins를 추가할 필요없이 설립 murine thymic T - 세포 림프종 라인의 개발을위한 세부적인 프로토콜을 제시한다. 종양은 hunched 자세의 관찰을 바탕으로 눈에 보이는 종양의 초기 표시에서 3~6개월 세 생쥐에서 수확 호흡, 가난한 정리하고 민감한 스트레인 1,4의 낭비를 보람 있었다. / - - [FVB/N- ATM tm1Led / J] 2 p53의 - / - [129/S6- Trp53 tm1Tyj / J] 5 쥐가 우리는 성공적으로이 프로토콜을 사용하여 여러 개의 T - 세포 라인과 ATM의 근친 변종을 설립했습니다. 우리는 더 이상 기존 T - 세포 인구의 90 % 이상이 인 경우에는 3 번 CD, CD4와 CD8을 표현 것을 보여줍니다. 안정 설립 세포 라인과 일치, 현재 프로토콜을 사용하여 발생하는 T - 세포는 일년 이상 passaged되었습니다.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

이 비디오에서는 마우스 thymic 림프종 세포 라인을 확립 프로토콜을 보여줍니다. 및 p53의 - / - - thymic 림프종과 생쥐이 프로토콜에 따라, 우리는 성공적으로 여러 ATM - /에서 T - 세포 라인을 설립했습니다.

에서 Thymic 림프종 T - 세포 라인의 유도 ATM - / -</sup과 p53의 - / -</sup 마우스

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K 조회수.  Cornell University. 이 비디오에서는 마우스 thymic 림프종 세포 라인을 확립 프로토콜을 보여줍니다. 및 p53의 - / - - thymic 림프종과 생쥐이 프로토콜에 따라, 우리는 성공적으로 여러 ATM - /에서 T - 세포 라인을 설립했습니다.

비디오: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

반대 미리 정의된 T 세포 수용체 Specificities와 Transnuclear 쥐 Toxoplasma gondii SCNT 비아 획득

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

연구

면역학 및 감염병학

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 ...

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 ...

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 ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T 세포의 유도<em&gt; 체외</em&gt; 마우스 배아 줄기 세포에서

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

골수 전구 세포의 레트로 바이러스 형질 도입은 T 세포 수용체 Retrogenic 마우스를 생성하는

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

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 ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK과 만성 활성 엡 스타인-바 바이러스 감염을 가진 환자에서 T 셀 라인을 설정 하는 효율적이 고 간단한 방법

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

에서 Thymic 림프종 T - 세포 라인의 유도 ATM - / -과 p53의 - / - 마우스

에서 Thymic 림프종 T - 세포 라인의 유도 ATM^{- / -과 p53의^{- / - 마우스}}