Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

이 연구는 교육, 싱가포르 교육부 (AcRF Tier1-RG40 / 13 Tier2-MOE2013 - T2-2-038)에 의해 지원됩니다. 원고는 Obrizus 통신에서 에이미 설리번에 의해 편집되었다.

모든 마우스는 사육 및 특정 병원균이없는 조건에서 유지되고 모든 마우스 프로토콜은 기관 동물 관리 및 사용위원회의 지침에 따라 실시하고있다. 버퍼 및 시약 1. 준비 <ol><li> 55 μM 2- 머 캅토 에탄올을 완료 로스웰 파크 메모리얼 연구소 (RPMI) 배지 (10 % 열 - 불 활성화 소 태아 혈청 (FBS), 2 mM L- 글루타민, 페니실린 (100 IU / ㎖) / 스트렙토 마이신 (100 μg의 / ㎖)을 제조 ). </li><li> 별도로, 20 배 균형 소금 용액 (BSS) 주식 1 재고 2를 준비합니다. <ol><li> 20 배 BSS의 1 개 (1 L 멸균 111 mM의 포도당, 8.8 MM의 인산 칼륨, 26.7 mM의 인산 나트륨)를 준비하고 최종 볼륨 조정하기 전에 20 배 BSS의 1 개에 40 ㎖의 0.5 % 페놀 레드를 추가합니다. 4 ℃에서 저장하기 전에 0.2 μm의 필터를 사용하여 살균 필터. </li><li> 20 배 스톡 BSS 2 (25.8 mM의 염화 칼슘 이수화 물, 107 mM의 염화칼륨, 2.73 M 나트륨 CH 준비loride 19.6 mM의 염화 마그네슘 육수화물, 1- L 멸균 16.6 mM의 마그네슘 설페이트). 4 ℃에서 저장하기 전에 0.2 μm의 필터를 사용하여 살균 필터. </li><li> 400 ml의 멸균 각각 별도로, 50 ㎖ BSS의 1 개 및 50 ml의 BSS 주식이 희석, 실험적인 사용을 위해 1X BSS를 준비합니다. 모두 희석 솔루션을 결합하여 pH 7로 조정하고, 20 ㎖의 FBS (2 %)를 추가합니다. 0.2 μm의 필터를 사용하여 멸균 및 살균 필터를 사용하여 1 L까지 최고. 참고 : 농축 BSS 주식 혼합 직접 침전이 발생할 수 있기 때문에 BSS의 재고 솔루션은 별도로 준비해야한다. </li></ol></li><li> 적혈구 (RBC) 용해 버퍼 <ol><li> , RBC 용해 버퍼를 준비 사용하기 전에 1 부품 재고 트리스베이스 (멸균 130 mM 트리스 (히드 록시 메틸) 아미노 메탄, 산도 7.65) 9 부에게 재고 암모늄 클로라이드 (멸균 155 MM의 염화 암모늄)를 혼합합니다. 참고 : 4 ° C에서 염화 암모늄 및 트리스베이스의 저장 멸균 재고 솔루션을 제공합니다. 용해 B를 제조적혈구의 효율적인 용해를 보장하기 위해 신선한 uffer. </li></ol></li></ol> 비장 또는 림프절에서 림프구 서스펜션의 2 세대 주 : 마우스를 안락사 이전 실험에 필요한 모든 시약 및 장치를 제조 및 높은 세포 생존율을 유지하기 위해 가능한 한 빨리 림프구의 단일 세포 현탁액을 생성하는 것이 중요하다. <ol><li> 자궁 경부 전위 또는 CO 2 질식에 의해 실험 마우스를 안락사. 참고 : 이후이 단계에서 모든 실험 절차는 무균 적으로 수행해야합니다. </li><li> 어떤 절개를하기 전에 70 % 에탄올로 전체 마우스를 찍어. 비장과 림프절 무균 8을 제거하고 또는 (단계 1.2.3에서) BSS / FBS (2 % FBS와 RPMI) 5 ml의 얼음처럼 차가운 RPMI / FBS를 포함하는 별도의 15 ml의 튜브에 배치합니다. 주 : BSS 효율적 RBC 용해를 방지하기 때문에, 비장 세포 현탁액의 제조 RPMI를 사용 afte BSS로 전환R RBC 용해. </li><li> 2 ml의 얼음처럼 차가운 RPMI / FBS 또는 BSS / FBS를 포함하는 페트리 접시에 멸균 100 μm의 셀 스트레이너 메쉬의 두 조각 사이에있는 기관 (들)을 배치, 비장이나 림프 노드에서 단일 세포 현탁액을 생성합니다. 그것은 매우 미세한 부분으로 찢어 때까지 1 ml의 주사기의 플런저를 사용하여, 기관 (들) 매쉬. </li><li> 15 ㎖의 튜브에 세포 현탁액을 전송하고, 셀 스트레이너 얼음처럼 차가운 RPMI / FBS 또는 BSS / FBS 메쉬 씻는다. 나머지 세포 현탁액을 수집 같은 15 ML 튜브에 추가하고, 4 ℃에서 5 분 동안 453 XG에 스핀 다운. 뜨는을 제거합니다. 주 : 다음 단계에서 RBC 용해 버퍼 또는 매체를 추가하기 전에 손가락으로 튜브를 flicking하여 펠렛 세포를 일시 중지 다시. </li><li> 세포 현탁액을 원심 분리 동안 실온 (RT) RBC 용해 버퍼를 준비한다 (단계 1.3 참조.). 상층 액을 세포를 펠렛 및 제거한 후, 매 10 8 세포를 1 ml의 RBC 용해 버퍼와 세포를 다시 일시 중지합니다. 인큐BATE 3-4 분 동안 RT에서 분해 반응. </li><li> 14 ml의 얼음처럼 차가운 BSS / FBS와 RBC 용해를 중지하고 4 ℃에서 5 분 동안 453 XG에 스핀 다운. </li></ol> B 및 T 세포의 정제 3. <ol><li> B 세포의 정제 <ol><li> 혈구 세포를 사용하여 계산. 300 ㎕의 BSS / FBS 최대 10 8 비장 세포를 다시 일시 중지하고 50 μl의 항 CD43에게 자기 마이크로 비드 9,10를 추가합니다. 죽은 세포를 제거 30 μl를 넥신 V 자석 구슬을 추가합니다. 30 분 동안 4 ℃로 냉장고에 세포 현탁액을 부화. </li></ol></li><li> T 세포의 정제 <ol><li> 혈구 세포를 사용하여 계산. 비 T 세포 고갈 항체 칵테일 10 8 세포 (CD19, B220, GR-1에 대한 비오틴 항체까지 다시 일시 중지, TCR-γδ, CD49b, 중 CD11c, CD11b를, Ter119 및 CD4 또는 CD8은에 표적 세포 인구에 따라 / FBS -4,5- 200 μL의 BSS 200 :) 정제 1 희석. A의 세포 현탁액을 부화15 분 동안 4 ° C 냉장고. </li><li> 배양 후, 세포를 세척하고 4 ℃에서 5 분 동안 453 XG에 스핀 다운 10 ml의 BSS / FBS를 추가합니다. 상층 액을 제거하고 30 μL의 스트렙 타비 딘 마이크로 비드 15 ㎕를 넥신 V 자기 구슬 165 μL의 BSS / FBS에서 세포를 다시 일시 중지합니다. 30 분 동안 4 ℃로 냉장고에 세포 현탁액을 부화. 주 : 심지어 자기 마이크로 비드와 라벨을 보장 15 분 동안 마이크로 비드와 세포를 배양 한 후 15 ML 튜브를 눌러 부드럽게 세포 현탁액을 혼합 단계 3.1.1 중에 다른 15 분을 품어. 또는 3.2.2. </li></ol></li><li> 셀 정화를위한 분리 컬럼의 제조 <ol><li> 세포의 마이크로 비드 라벨 동안 사용되지 않은 분리 컬럼을 준비한다 (단계 3.1.1. 또는 3.2.2.). RT에 (FBS)없이 사전 따뜻한 BSS 및 세척 및 무균 열 평형을 2 ml를 사용합니다. BSS와 평형 후, 세척 된 열은 건조하도록 허용해서는 안된다. 참고 : 우리는 LS의 C를 사용하여olumn 대신 권장 LD 열 인해 재사용 성을 (단계 3.4를 참조하십시오.). </li><li> 자기 구슬 표시 한 후, 세포를 세척하고 4 ℃에서 5 분 동안 453 XG에 스핀 다운 14 ml의 BSS / FBS를 추가합니다. 상층 액을 제거하고 1-3 ml의 RT BSS에서 세포를 다시 일시 중지합니다. </li><li> 정제 과정에서 유속을 감소시키기 위해 열 선단에 멸균 21 G 바늘을 부착. 평형화 컬럼에 세포 현탁액을 넣고 정제 된 표적 세포를 함유 통해 흐름을 수집한다. 주 : 열로드하는 동안에 거품을 도입하지 마십시오. </li><li> 1 ml의 BSS / FBS 한 번 컬럼을 세척하고 정제 된 표적 세포를 포함 통해 흐름을 수집합니다. 다시 한번를 통해 흐름에 열을 다시로드합니다. 동일한 15ml를 튜브에 제 2 로딩 돌파 흐름을 수집한다. </li><li> 1 ml의 BSS / FBS와 열을 3 회 세척하고 정제 된 표적 세포를 포함 통해 흐름을 수집합니다. 그 후, 토륨 5 ml의 BSS / FBS를 추가플런저와 전자 칼럼은, 새로운 15 ML 튜브에 열을 밖으로 자기 표지 된 세포를 세척합니다. </li><li> 정제 B 또는 T 세포의 항원 4,5- 표면에 결합하는 항체를 사용하여 유동 세포 계측법에 의해 수집 된 세포의 순도를 확인한다. </li></ol></li><li> 다시 사용하여 분리 컬럼을 주 : LS 열이 정화 효율에 영향을주지 않고 4 회 이상 재사용 할 수있다. <ol><li> 5 ml의 포스페이트 완충 식염수 (PBS)와, 플런저를 사용하여 상부로부터 증류수 5 mL로 3 회 컬럼을 3 회 세척 하였다. </li><li> 5 ml의 70 % 에탄올로 컬럼을 세척하고 광범위 열에 녹의 축적을 방지하기 위해 공기 탭을 사용하여 열을 건조. </li><li> 별도의 정화 실험을 위해 사용 된 LS 열을 준비하려면, 주사기 어댑터를 사용하여 5 ml의 70 % 에탄올로 아래에서 위로 열을 씻는다. 다음으로, 컬럼의 상단에서 한 번에 5 ml의 PBS 다음에 5 ml의 멸균 PBS로 두 번 아래에서 위로 열을 씻는다. 2 추가ml의 RT BSS는 열 평형을 다음 라벨 세포와 열을로드로 진행합니다. </li></ol></li></ol> 4. CFSE 라벨 및 자극 주 : 정제 된 세포는 시험 관내의 다양한 생체 기능 분석에서 실시 될 수있다. 여기서는 장갑차 (5)의 T 세포 자극 기능을 결정하기 위해 정제 된 T 세포를 사용한다. <ol><li> 37 ° C 이전 CFSE에 로딩 사전 따뜻한 라벨 솔루션 (PBS에서 0.1 % FBS). 참고 : PBS에서 FBS의 낮은 비율을 사용하여이 CFSE 로딩 중 세포 사멸을 감소시키고 원심 분리하는 동안 세포 손실을 최소화 할 수 있습니다. 그러나, 너무 많은 FBS는 CFSE 로딩을 방해 할 수 있습니다. </li><li> 라벨 용액으로 두 번 정제하여 세포를 세척 한 다음 15 ㎖의 튜브 예열 라벨링 용액에 2 × 107 세포 / ㎖로 재현 탁. </li><li> 10 μM CFSE의 용액을 제조 하였다 (1 : 500 희석 5mM의 CFSE 원액) 예열 라벨 솔루션이다. CFSE 솔루션해야갓 최적 라벨링을 달성하기마다 준비. </li><li> CFSE로 세포를 로딩하기 위해, 37 ° C에서 10 분 동안 어두운 데에서 15 ㎖의 튜브에 1 부분 10 μM CFSE의 용액에 한 부분에 세포 현탁액을 추가 배양한다. 5 μM CFSE의 최종 농도는 1 × 10 7 세포 / mL의 라벨을 사용한다. </li><li> CFSE 로딩 동안 세포 균질 한 혼합을 보장하기 위해 관마다 2 분 전환. </li><li> 반응을 중지 얼음처럼 차가운 완전한 RPMI 매체의 여러 볼륨을 추가, 4 ° C에서 5 분 동안 453 XG에 스핀 다운합니다. 성공적인 CFSE 로딩되면, 세포 펠렛은 황색 표시됩니다. </li><li> 워시 CFSE는 얼음처럼 차가운 완전한 RPMI 배지로 세포를 한 번 더로드 및 체외 배양 또는 생체 자극에 사용하기 전에 4 ° C에서 5 분 동안 453 XG에 스핀 다운. </li></ol> 5. 체외 자극 <ol><li> 바로 사용하기 전에 자극의 2 배 원액 (2 배 자극 원액)를 준비 (10) 그래서배 자극 원액 0 μL를 96 웰 플레이트에 웰 당 200 ㎕의 최종 부피로 세포 100 μl를 첨가 할 수있다. </li><li> (B 세포 또는 T 세포 CD3 및 CD28 또는 CD40에 대한 IgM의) 자극 코팅 플레이트가 필요할 경우, 밤새 4 ℃에서 PBS에 자극 및 프리 코트 배양 접시를 희석. 대안으로, 배양 용기는 실험 하루에 1 시간 동안 37 ℃에서 코팅 할 수있다. PBS (접시 언제든지 건조하는 것을 허용하지 않는다)로 두 번 코팅 된 판을 씻으십시오. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> B 세포 </td><td></td></tr><tr><td> 자극 </td><td> 최종 농도 </td></tr><tr><td> F (AB &#39;) 2 염소 항 - 마우스 IgM의 </td><td> 0.6-2.4 μg의 / ㎖ </td></tr><tr><td> 반대로 마우스 CD40 단클론 항체 </td><td> 0.5 μg의 / ㎖ </td></tr><tr><td> 재조합마우스 IL-4 </td><td> 25 U / ㎖ </td></tr><tr><td> 리포 폴리 사카 라이드 </td><td rowspan="2"> 0.1 μg의 / ㎖ </td></tr><tr><td> E.에서 (LPS) 대장균 혈청 형 055 : B5 </td></tr><tr><td></td><td></td></tr><tr><td> T 세포 </td><td></td></tr><tr><td> 자극 </td><td> 최종 농도 </td></tr><tr><td> 항 CD3 (플레이트 코팅) </td><td> 2-10 μg의 / ㎖ </td></tr><tr><td> (50 μL / 웰 코팅) </td><td></td></tr><tr><td> 안티 CD28 (플레이트 코팅) </td><td> 2 μg의 / ㎖ </td></tr><tr><td> 재조합 IL-2 </td><td> 40 U / ㎖ </td></tr><tr><td> PDBu (포르 볼 에스테르) </td><td> 5-50 ng를 / ㎖ </td></tr><tr><td> A23187 (칼슘 이온 운반체) </td><td> ng를 250 / ㎖ </td></tr></tbody></table> 표 1 : 체외 배양에서 림프구를 자극하는 데 사용되는 자극의 농도.<ol start="3"><li> CFSE 표지 된 B 세포의 경우, 3 × 105 세포 중으로의 완전한 RPMI 배지 및 배양 6 × 10 3 세포 / ml을 다시 중지 / 웰로 72 시간 동안 96- 웰 편평 바닥 플레이트. </li><li> CFSE 표지 된 T 세포, 0.5~3 × 105 세포 중으로의 완전한 RPMI 배지 및 배양에 0.5~3 × 106 세포 / ㎖로 재 - 정지 / 웰에서 48 또는 72 시간 동안 96 웰 둥근 바닥 플레이트 . </li></ol> 6. 생체 자극 <ol><li> 생체 자극, 적응 적 전송을위한 마우스 당 4 × 10 6 CFSE 표지 T 세포 (정맥 주사 (IV) 200 ㎕의 PBS에) 각 MHC-일치받는 사람 마우스로. 참고 :이 프로토콜에서, CFSE 표지 T 세포가 적응 적 집 같은 비장과 림프절 등 장기 림프하는 것입니다 이러한 세포로 꼬리 정맥 또는 역 궤도 주사를 이용하여 전송할 수 있습니다. </li><li> 일일 나중에 항원받는 사람 마우스에 도전. 노트:OVA 특이 적, T 세포 수용체 (TCR) -transgenic T 세포가 적응 적받는 마우스에 옮겼다 때문에이 예에서는 항원으로서 난 알부민 (OVA 단백질를 50㎍ / 마우스)를 사용한다. 각받는 사람 마우스 (5)로, 피하 주사 (SI)를 통해 멸균 PBS에서 OVA 단백질을 준비하고 OVA 단백질 / PBS 또는 PBS 제어의 100 μl를 주입. </li><li> 수확 3 일 OVA 단백질 또는 PBS와 예방 접종 후받는 사람 마우스의 단일 림프 기관에서 세포 현탁액 (림프절과 비장)을 생성합니다. 근위 림프절 장간막, 오금, 사타구니, 허리와 꼬리 림프절을 포함 겨드랑이, 상완 및 표면 경부 림프절) 및 말초 림프절 (DLN)를 포함한다 (PLN),에 별도의 림프절. FACS 적절한 항체를 사용하여 염색 된 세포는 T 세포의 증식을 확인한다. 주 :이 CFSE 셀 추적 실험에서 비 디위한 기준선 형광을 설정 PBS가 주입 된 대조군 마우스로부터 T 세포를 CFSE 표지세포를 이용하므로. 증식 세포 분열은 항원 자극, CFSE 표지 된 세포는 형광 피크 (12, 13)을 측정함으로써 가시화된다. PBS를 제어으로 증식 세포 분열의 수, CFSE 표지 된 T 세포 12,13 결정될 수있다. </li><li> 샘플 사이의 세포 분열이나 피크의 수를 비교하여 CFSE 표지 된 세포 증식 데이터를 분석 (도 1 및도 2). 참고 : 예를 들어, CFSE 표지 된, 적응 적 난자 항원을 수신받는 마우스에 전달 OVA 특이 적 T 세포는 PBS가 주입 된 대조군 마우스에 비해 5,12- 활성 증식을 받게 될 것이다. 또한, 호킨스 동료 (14)에 의해 입증 된 바와 같이 CFSE 세포 증식 데이터를 분석하는 다양한 방법이 있다는 것을 지적 주목할 만하다. </li></ol>

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The authors have nothing to disclose.

이 프로토콜에서는, 우리는 림프 기관에서 림프구를 정제하는 과정을 보여줍니다. 자기 비드 정렬을 사용하여 셀 정화가 가능한 고도로 정제 된 표적 세포를 산출 빠르고 간단한 방법이다. 프로토콜 내에서 중요한 단계 세포 생존 및 세포 수율 체외에서 조혈 계통 세포의 생존을 유지하는 것은 성공적인 및 재현 ?...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 (without L-Glutamine)</td><td>Gibco</td><td>31870025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td>Heat inactivated&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030024</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140114</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-CD43 magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-049-801</td><td>Mix well prior use</td></tr><tr><td>Streptavidin microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td>Mix well prior use</td></tr><tr><td>Anti-Annexin V magnetic beads</td><td>Miltenyi Biotec</td><td>130-090-201</td><td>Mix well prior use</td></tr><tr><td>MACS LD&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>96-well U-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>650180</td><td></td></tr><tr><td>96-well F-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>655180</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; sterile disposable filter units</td><td>Nalgene</td><td>567-0020</td><td>Can be substituted with any sterile filter device</td></tr><tr><td>CellTrace Violet</td><td>Invitrogen</td><td>C34557</td><td>CTV for short; alternative to CFSE</td></tr><tr><td>CellTrace Yellow</td><td>Invitrogen</td><td>C34567</td><td>CTY for short; alternative to CFSE</td></tr><tr><td>CellTrace Far Red</td><td>Invitrogen</td><td>C34564</td><td>CTFR for short; alternative to CFSE</td></tr><tr><td>Cell Proliferation Dye eFluor 670</td><td>eBioscience</td><td>65-0840</td><td>CPD670 for short; alternative to CFSE</td></tr><tr><td>PKH26</td><td>Sigma Aldrich</td><td>PKH26GL</td><td>PKH26, alternative to CFSE</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>S7653</td><td>Can use from other sources</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma Aldrich</td><td>M2393</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Tris-base</td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8418</td><td></td></tr><tr><td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>Molecular Probes</td><td>C-1157</td><td>Reconstitute in DMSO</td></tr><tr><td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td><td>Sigma Aldrich</td><td>P1269</td><td></td></tr><tr><td>A23187 (Calcium ionophore)</td><td>Sigma Aldrich</td><td>C7522</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b biotin (clone m1/70)</td><td>Biolegend</td><td>101204</td><td>T cell depletion cocktail</td></tr><tr><td>CD11c biotin (clone N418)</td><td>Biolegend</td><td>117304</td><td>T cell depletion cocktail</td></tr><tr><td>Gr-1 biotin (clone RB6-8C5)</td><td>Biolegend</td><td>108404</td><td>T cell depletion cocktail</td></tr><tr><td>Ter119 biotin (clone Ter119)</td><td>Biolegend</td><td>116204</td><td>T cell depletion cocktail</td></tr><tr><td>TCR-&amp;gamma;&amp;delta; biotin (clone GL-3)</td><td>Biolegend</td><td>118103</td><td>T cell depletion cocktail</td></tr><tr><td>CD19 biotin (clone 6D5)</td><td>Biolegend</td><td>115504</td><td>T cell depletion cocktail</td></tr><tr><td>B220 biotin (clone RA3-6B2)</td><td>Biolegend</td><td>103204</td><td>T cell depletion cocktail</td></tr><tr><td>CD49b biotin (clone DX5)</td><td>Biolegend</td><td>108904</td><td>T cell depletion cocktail</td></tr><tr><td>CD4 biotin (clone GK1.5)</td><td>Biolegend</td><td>100404</td><td>T cell depletion cocktail</td></tr><tr><td>CD8 biotin (clone 53-6.7)</td><td>Biolegend</td><td>100704</td><td>T cell depletion cocktail</td></tr><tr><td>F(ab&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</td><td>Cyt-282</td><td></td></tr><tr><td>LPS from E. coli Serotype 055:B5</td><td>Sigma Aldrich</td><td>L-4005</td><td></td></tr><tr><td>Anti-CD3 (clone clone OKT3) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-2</td><td>ProSpec</td><td>Cyt-370</td><td></td></tr><tr><td>Albumin from chicken egg white, Ovalbumin</td><td>Sigma Aldrich</td><td>A7641</td><td></td></tr></tbody></table>

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.

림프구 세포 자기 정화 사용자가 상대적으로 짧은 시간 동안 표적 세포 집단을 정제 할 수있다. 우리 공핍 프로토콜을 사용하여, 정제 후 94.2 % (에 72.8 % (전 정제) 행 (재결합 활성화 유전자 1 (RAG-1) 결핍 생쥐 OT-I) CD8 T 세포의 비율을 증가 할 수 있었다; 도 1a) 4,5. 이러한 정제 림프구는 림프구 증식 및 신호 전달 4,5-을 결정하는 다운 스트림 기능...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

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.

분리 및 쥐 림프구의 활성화

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

isolation-and-activation-of-murine-lymphocytes

Research

JoVE Journal

Immunology and Infection

21.0K 조회수.  Nanyang Technological University. 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.

비디오: Isolation and Activation of Murine Lymphocytes

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

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 ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

마우스 Monocyte 파생 된 모 수석 세포 및 종양 면역 단지와 활성화 후속 생체 외에서 그들의 격리 프로토콜

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

암 연구학

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4의 분리 + Miltenyi 맥을 정제를 사용하여 마우스 림프절에서 T 세포

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

생물학

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

마우스 성기 요로 점막에서 림프구의 분리

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

마우스 수지상 세포 하위 집합의 분리를위한 효율적이고 높은 수율 방법

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

마우스 작은 장의 면역 시스템에서 세포를 분리

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Murine Dermal Lymphatic Capillaries에서 내피 세포 분리

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

저자 스포트라이트: Magnetic Bead-Based Isolation of murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Murine Dermal Lymphatic Endothelial Cell Isolation(Murine 피부 림프 내피 세포 분리)

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...