This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

이 프로토콜은 인간의 환자 물질로부터 림프 세포의 분리를 설명하고, 따라서 윤리적 승인을 필요로한다. 본 연구에서 수행 된 작업은 웁살라 윤리 심의위원회 (DNR. 387분의 2,013)에 의해 부여했다. 인간 팔라티노 편도선에서 단핵 세포 (다국적 기업) 1. 분리 주의 : 혈액, 조직 또는 체액과 같은 인간 기원의 모든 차폐 재료는 잠재적 감염 물질로 간주되어야한다. 따라서, 인간의 조직을 처리하기위한 권장 바이오 안전성 관행에 따라야한다. 참고 : 오염을 방지하기 위해 모든 솔루션 및 세포 배양 장비는 멸균해야합니다. 모든 버퍼와 솔루션은 사전 냉각 얼음에 보관해야합니다. 편도 조직과 분리 된 세포는 보관하고 얼음에 처리되어야한다. <ol><li> 편도 또는 tonsillotomy (그림 2)을받은 환자에서 신선한 편도선을 얻습니다. 조직 clum 플 런지5 % 소 태아 혈청 (FBS), 10 mM의 글루타민, 0.05 밀리그램 / 젠타 마이신 및 1 % 항생제 - 안티 곰팡이 믹스 ㎖ (페니실린 빙냉 멸균 된 행크스 평형 염 용액 (HBSS)으로 가득 50 ㎖ 원심 분리 관에 추신 스트렙토 마이신 및 암포 테리 신 B). </li><li> 얼음에 항상 잠겨 편도 조직 샘플 튜브를 유지한다. 가능한 빨리 편도선을 처리하려고없고 나중에 제거 수술 후 3 시간 이상. </li><li> 얼음에 60mm 플라스틱 세포 배양 접시에 편도선을 놓고 HBSS에 적신 조직을 유지. 편도선 표면에서 깨끗한 집게로 볼 혈전, 지방과 결합 조직을 제거합니다. </li><li> 5 ㎖의 HBSS를 함유하는 새로운 60mm 세포 배양 플레이트를 사용한다. 가위 및 / 또는 메스의 멸균 쌍을 사용하여 3~10mm의 조각으로 편도선 조직 덩어리를 잘라. </li><li> HBSS 10 ㎖를 함유하는 새로운 60mm 세포 배양 플레이트를 준비하고, HBSS 용액에 100㎛의 플라스틱 셀 스트레이너를 배치했다. </li><li> DISS 이동멸균 집게와 셀 스트레이너에 반사된다 편도 조각. 플라스틱 주사기의 플런저의 단부를 사용하여 부드럽게 셀 스트레이너를 통하여 상기 조직 절편을 짠다. 편도 조각이 완전히 HBSS에 몰입되어 있는지 확인합니다. </li><li> 티슈 남아있는 셀 스트레이너를 폐기하고 50 ㎖ 플라스틱 원심 분리 튜브에 세포를 60mm 배양 접시에서 얻어진 세포 현탁액을 전송. 단계 1.8 및 1.9를 진행하는 동안 얼음에 튜브를 남겨주세요. </li><li> 편도선 큰 경우, 크기가보다 큰 2cm (도 2), 스트레이너 메쉬에서의 막힘을 방지하기 위해 두 개의 셀 스트레이너에 편도선 단편을 짠다. </li><li> 세포 현탁액 35 ml를 최종 부피를 얻었다. 주 :이 단계에서 그것은 동결 보존 (섹션 2 참조) 세포 현탁액을 제조 할 수있다. </li><li> 새로운 50 ㎖ 원심 분리 튜브에 피콜 같은 밀도 구배 용액 10 ㎖를 추가한다. 조심스럽게 세포 suspens 오버레이이온 밀도 구배 용액의 상단에 (단계 1.9). 세포 현탁액 및 밀도 구배 용액의 혼합을 피한다. 주 : 밀도 구배 용액을 RT로 가온되어야한다. </li><li> 실온에서 20 분 동안 700 XG에서 스윙 아웃 로터의 세포 현탁액을 원심 분리기. 그라데이션을 방해 할 수 있습니다 빠른 제동과 원심 분리기에 브레이크 기능을 활성화하지 마십시오. 또한, 원심 분리기에서 사용할 수있는 가장 낮은 가속 기능을 사용합니다. 주 :이 원심 분리 단계 이후에, 상기 튜브의 하단에 퇴적물로서 계면에서 무성 백색 층, 및 적혈구, 섬유 아세포와 같은 세포 파편 다국적 관찰. </li><li> 조심스럽게 10 ㎖ 피펫을 사용하여 다국적 층을 수집한다. 새로운 50 ML의 원심 분리기 튜브에 세포 현탁액을 놓습니다. </li><li> 세포 현탁액에 얼음처럼 차가운 PBS의 25 ML을 추가하고 4 ℃에서 스윙 아웃 회에서 5 분 동안 300 XG에 튜브를 회전. </li><li> 25 ML의 피펫을 이용하여 상층 액을 제거세포 펠렛 세척 단계를 두 번 더 반복한다. 마지막으로 15 ml의 PBS에서 세포 펠렛을 재현 탁. 주 : 최종 세척 단계 후에, 세포 현탁액을 (섹션 2 참조) cryopreserve하는 것이 가능하다. </li><li> 혈구 세포 계수 챔버에 세포 현탁액 10 μL를 적용하고 현미경으로 세포 수를 계산. 세포의 적절한 양을 분배 (예, 2-3 X 107) 이후의 자성 비드의 분리를 위해 15 ml의 원심 분리 튜브에. 단계 3.2까지 얼음이 나누어 보관하십시오. </li></ol> 편도 세포의 2. 냉동 보존 <ol><li> 냉동 매체 (90 % FBS 10 % DMSO)를 준비하고 얼음에 보관하십시오. 장기간 저장을 위해 -20 ℃에서 냉동 보관 매체. </li><li> 혈구 세포 계수 챔버 (단계 1.15)를 이용하여 세포의 수를 결정한다. 냉동 원하는 세포 밀도에 따른 동결 배지의 요구량을 계산한다 (예를 들어, 107 셀S 동결 배지 / ㎖). </li><li> 5 분 동안 300 XG에 세포 현탁액을 원심 분리기. 세포 펠렛을 방해하지 않고 상등액을 경사 분리하고 (단계 2.1) 빙냉 동결 배지에서 세포 펠렛을 재현 탁. </li><li> 액체 질소에 장기 저장을 위해 설계 멸균 바이알에 세포 현탁액을 1 ml의 분취 량을 분배. 이소프로판올 실에서 튜브를 동결 및 -80 ° CO / N에 저장합니다. 장기 보존을위한 저장 탱크 또는 -140 ° C 세포를 함유하는 냉동 액체 질소로 튜브를 옮긴다. </li><li> 냉동 세포와 튜브를 해동, 37 ° C의 물을 욕조에 빠르게 그들을 따뜻하게. 해동시 바로, (보충제 단계 1.1) 예열 된 HBSS 10 ㎖에 균체 현탁액을 분산 15ml를 원심 분리 관에. (단계 1.1, 보충제), 5 분 동안 300 XG에 스핀 HBSS 튜브를 제거하고 HBSS에서 원하는 세포 밀도에서 세포 펠렛을 재현 탁. 참고 : 다국적 기업의 AF의 가능성을터 해동은 사균의 존재 정제 된 B 및 T 림프구의 최종 수율이 감소하기 때문에, 중요하다. 주 : 세포 분리 효율을 감소시킬 것이다 세포 생존율이 80 % 이하 후 경험하는 방법. </li></ol> 편도 다국적 기업에서 T 림프구 인구 3. 긍정적 인 선택 주 1 :이 프로토콜은 CD3 항체에 결합 된 자성 비드를 이용하여 편도 다국적로부터 인간 CD3 + T 림프구의 포지티브 선택에 기초한다. 그것은 신선한 (1 절) 또는 냉동 (제 2) 다국적 기업에서이 부분을 시작하는 것이 가능하다. 주 2 : 3 × 10 7 다국적 기업과 함께 시작합니다. 분리 컬럼은 막힐 수 있고, 이는 분리 효율을 줄일 수 있기 때문에,이 세포의 수를 초과하지 않는다. 이상의 셀이 처리 될 경우 더 큰 열을 사용합니다. 이 프로토콜에서 사용되는 양을 실험적 우리 세포의 수에 대해 최적화 된우리의 실험 설정에서 에드. <ol><li> 분리 완충액 [PBS (PH 7.2), 0.5 % 소 혈청 알부민 (BSA) 및 2 mM EDTA]를 준비한다. 4 ° C에서 0.45 μm의 필터와 저장소를 통해 분리 버퍼를 필터링합니다. </li><li> 4 ℃에서 5 분 동안 300 XG에 다국적 기업 서스펜션 (단계 1.14)를 스핀. 빙냉 분리 완충액 240 μL로 얻어진 세포 펠렛 (107 세포 당 80 μL)을 재현 탁. 멸균 2 ML 튜브에 세포 현탁액을 전송합니다. </li><li> 셀 솔루션 CD3 자기 항체의 20 μl를 추가합니다. 서스펜션에 세포를 유지하기 위해 지속적으로 부드러운 혼합하여 4 ℃에서 1 시간 동안 튜브를 품어. </li><li> 세포를 세척하기 위해 5 ㎖ 빙냉 분리 완충액을 함유하는 15 ㎖의 튜브에 세포 현탁액을 모두 전송할. </li><li> 스윙 아웃 회에서 4 ° C에서 10 분 동안 300 XG에 튜브를 원심 분리기. </li><li> 한편 자기 분리기 및 열을 설정합니다. 스탠드에 자기 분리기를 부착하고 separ에 열을 배치ATOR. 빙냉 분리 완충액 500 μL를 적용하여 컬럼을 세척 하였다. 을 통해 흐름을 폐기하십시오. </li><li> 열 아래에 새 15 ml의 수집 튜브를 놓습니다. 얼음에 포집 관을 유지합니다. </li><li> 피펫으로 상층 액 (단계 3.5)을 폐기하고 부드럽게 500 μL 분리 버퍼에서 세포 펠렛을 재현 탁. 사전 세척 칼럼의 상단에있는 세포 현탁액을 적용하고 그것을 통해 실행하자. 주 : 세포 덩어리가 열을 막히게함으로써 유량을 감소시킬 수있다. 이 문제를 방지하기 위하여, 컬럼의 상단에 40 μm의 플라스틱 셀 스트레이너를 배치했다. 이 메쉬를 통해 셀을 통과하는 고도로이 방법의 효율성을 향상시킬 것이다. </li><li> 분리 버퍼의 1.5 ㎖ (10 7 세포 500 μL)으로 열을 4 회 반복한다. 다음 세척 단계를 적용하기 전에 (어떤 액체가 열에서 관찰 될 수 없습니다) 비어있는 열 저장 기다립니다. 주 : B 림프구 분획으로서 간주 CD3 표지 된 세포를 얻는, 그들이 그대로포집 관에 용출. </li><li> 새로운 15 ml의 수집 관에 분리 및 장소에서 열을 제거합니다. 피펫 컬럼 상 분리 완충액 2 ㎖. T 림프구의 비율로 간주된다 긍정적 선택된 T 림프구를 용출 컬럼 공급 플런저 사용. </li><li> 정제 세포 (단계 1.15) 필요한 경우의 수를 결정합니다. 세포 현탁액은 지금 하류 실험에 대한 준비가되어 있습니다. </li></ol> 절연 4. 유동 세포 계측법 분석 편도 B와 T 림프구 주의 : 파라 포름 알데히드 용액을 자극 의심되는 발암 물질이다. 적절한 보호 복, 보호 장갑과 눈 / 안면 보호구를 착용 할 것. 주 1 :이 프로토콜은 (FACS) 분석 정리 형광 활성화 된 세포에 의해 격리 된 B 및 T 세포의 염색을위한 직접적인 방법을 설명한다. 이 단계의 주요 목적은 인구 AF를 격리 세포의 순도를 평가하는 것이다터 CD3 자기 항체 분리. 이 목적을 확립 B 및 T 세포 마커에 대해 형광 공역 모노클로 날 항체 및 CD20-FITC CD2-APC가 사용된다. 또한 이소 제어 항체 포함 높게 CD2 및 CD20 항체 (단계 4.8 참조)의 결합 비특이적 &quot;배경&quot;을 구별 할 것을 권장한다. 2 주 : 염색 할 때까지 4 ° C에서 1 % 파라 포름 알데히드에 어둠 속에서 (PFA) 솔루션을 정제 된 세포 분획을 유지할 수있다 (예를 들면, 다음 날.). 염색 및 FACS 분석 간의 시간 간격이있는 경우에도 동일한 방법을 적용한다. 두 경우 모두에서 세포가 PBSA 염색 또는 FACS 분석하기 전에 버퍼 (PBS가 0.2 % BSA를 포함) 제대로 세척해야 함을 기억하십시오. <ol><li> 단계 3.10에서 단계 3.9와 T 림프구 분획에서, B 림프구 분율 (이전 컬럼에 적용하기) 단계 1.15에서 다국적 기업의 6 × 10 6 세포를 꺼내. </li><li> 세포를 스핀스윙 아웃 로터를 사용하여 4 ℃에서 5 분 300 XG에 현탁액. </li><li> 피펫으로 상층 액을 제거하고 얼음 - 냉각 PBSA 1 ㎖로 한번 세포 펠렛을 세척 하였다. 4 ℃에서 5 분 동안 300 XG에 세포 현탁액을 스핀. 피펫으로 상층 액을 제거하고를 재현 탁 얼음처럼 차가운 PBSA 버퍼 600 μL 세포. </li><li> FACS 튜브의 바닥에 세포 현탁액 100 ㎕를 추가한다. </li><li> PBS는 각 튜브에 10 %의 열 불 활성화 된 인간 혈청을 포함하는 100 μl를 추가, 잘 섞어 ~ 실온에서 1 분 또는 얼음에 20 분 동안 품어. 참고 : B 림프구가 된 Fc 수용체를 실시한다. 된 Fc를 차단하기 위해 정제 된 세포 분획을 10 % 열 불 활성화 인간 혈청과 함께 인큐베이션 수용체. 인간 혈청을 1 시간 동안 56 ℃에서 배양함으로써 열 불 활성화이다. -20 ℃에서 냉동 작은 분량 씩 저장에 열 불 활성화 혈청을 나눈다. </li><li> 자상 한 4 ℃에서 5 분 동안 300 XG에 세포 현탁액을 원심 분리기보내고 아웃 로터. </li><li> 피펫으로 상층 액을 제거하고 1 ml의 PBSA와 세포 펠렛을 씻는다. 원심 분리 (4 ℃에서 5 분 300 XG)를 반복합니다. </li><li> 얼음에 각각의 튜브에 100 μL PBSA를 추가합니다. 제조자의 권고에 따라, 형광 공역 단클론 항체의 적절한 양을 추가 (예컨대, 항 -CD20 20 μL 및 / 또는 PBSA 100 ㎕의 항 - CD2 항체의 5 μL). 참고 : FACS 분석을위한 충분한 제어 튜브를 할당하는 것을 잊지 마십시오. 이 프로토콜에서 다음 컨트롤 포함한다 : 개별적 안티 CD2 및 항 CD20 항체로 염색 1) 세포를 첨가하지 않은 개별 안티 CD2 및 항 CD20 아이소 타입 컨트롤 항체 및, 3) 세포 염색 2) 세포 항체. </li><li> 간단히 튜브 와동과 어둠 속에서 4 ° C에서 30 분 동안 품어. </li><li> PBSA와 함께하는 과정을 2 회 반복한다. 샘플 1 % PFA 용액 2 ㎖를 추가합니다. 셀 4에서 PFA 용액에 남아 있도록76; FACS 분석시까지 어두운 C. </li><li> 즉시 외과 기계에서 샘플을 실행하기 전에, (4 ℃에서 5 분 동안 300 XG)를 PBSA와 셀을 2 회 반복한다. 1 ㎖의 PBS와에 재현 탁 샘플은 이전의 흐름 cytometer의 분리, 빛으로부터 보호, 4 ° C (또는 얼음)하시오. </li><li> 제조업체 흐름 cytometer에서 프로토콜 다음 FACS 정렬 작업을 수행합니다. </li></ol> 고립 된 편도 B와 T 림프구에서 아데노 바이러스의 DNA 5. PCR 검출 주 1 : 아데노 hexon 유전자 AdRJC1 프라이머 (5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;)이고 AdRJC2 (5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;) 11. 이 프라이머는 139 BP의 증폭 물을 생성한다. 호스트 18S rRNA 유전자는 프라이머 tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;) 및 tp207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;)로 검출 할 수있다. 예상 amplicon의 크기는 300 BP이다. 주 2 : 모든 PCR실행은 음성 대조군 (증류수 H 2 O) 및 인간 아데노 바이러스 5 형 (12)에 감염된 인간 B 세포주 (BJAB)로부터 얻어지는 포지티브 컨트롤 DNA를 포함한다. <ol><li> 실리카 막 기반 열 정화 방법을 사용하여 (최소 1 × 10 6 세포 단계 3.11에서) DNA를 추출합니다. 페놀 및 구아니딘 이소 티오 시아 네이트 솔루션 (13)를 사용하여 RNA를 추출합니다. </li><li> 1X HF 완충액, 데 옥시 뉴클레오티드 트리 포스페이트 믹스 0.2 밀리미터 (의 dNTP), 0.25 각 프라이머 μM 및 20 ㎕의 총 부피에서 고성능 DNA 중합 효소 1 U를 함유하는 반응 혼합물 B 및 T 림프구로부터 100ng의 DNA를 추가한다. </li><li> 다음 사이클의 조건에서 PCR 증폭을 수행 10 초, 67 ° C (hexon) 또는 58 ° C (18S rRNA의) 30 초 72 ° C 98 ° C의 30주기 다음 98 ℃에서 30 초 변성, 30 초 동안. PCR 반응은 72 ℃에서 7 분의 최종 신장 단계를 종료 하였다. </li><li> 결과의 PCR 충전 앰프를 분리1X TBE에서 1.5 % 아가 로스 겔상에서 주시죠 제품 버퍼 핵산 염색으로 염색하여 예상 밴드를 시각화. </li></ol>

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The authors have nothing to disclose.

이 프로토콜의 결과에 영향을 미치는 가장 중요한 인자 중의 하나는 출발 물질로서 신선한 편도 물질의 사용이다. 따라서, 편도선 샘플은 수술 후 3 시간 이내에 처리해야한다. 편도선은 어른과 어린이 모두에서 얻을 수있다. 어린이에서 편도 물질로 인해 편도선 (tonsillotomy)의 부분 제거 수술에 일반적으로 작다. 따라서, 다국적의 수가 적은 편도선 샘플 (1 × 10 8 -2 × 109)과 비교된?...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

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			<td>Hanks balanced salt solution (HBSS)&#160;</td>
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			<td></td>
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			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
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			<td>Freezing medium</td>
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			<td>90% FBS and 10% DMSO</td>
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			<td>MACS buffer</td>
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			<td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td>
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			<td>PBSA&#160;</td>
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			<td>Fisher Scientific</td>
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			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
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			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
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			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
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			<td>40&#956;m plastic cell strainers&#160;</td>
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			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
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			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
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			<td>MACS MS columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
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			<td>MACS human CD3 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-881</td>
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			<td>Miltenyi Biotec</td>
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			<td>Merck Millipore</td>
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			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
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			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
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			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
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			<td>Human serum&#160;</td>
			<td>Rockland Immunochemicals</td>
			<td>D119-0100</td>
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			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-530L</td>
			<td></td>
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			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352003</td>
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			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72379</td>
			<td></td>
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			<td>BD LSRII flowcytometer</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
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			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
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			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
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			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
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			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
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			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</td>
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efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

B와 T 림프구의 고순도 소집단에서 편도 다국적 결과의 효율적인 분리. 이는 B 및 T 림프구 집단 각각 (도 3a)을 검출하기 위해 항 -CD20 및 항 CD2 항체를 사용하여 FACS 분석에 의해 확인 하였다. 한편, B 및 T 림프구 아 집단 (도 3b) 모두에서 세포의 혼합물이다 세포 분획 다국적 결과 비효율적 분리. 절연 편도의 B 및 T 림프구 (도 3A) 핵산 분리 ...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

인간 팔라티노 편도선에서 B와 T 림프구에 대한 효율적인 분리 프로토콜

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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15.7K 조회수.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

비디오: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

마이 그 레이션의 인간 T 림프구 분리, 문화 및 분석 체외에서</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

탯줄 혈액에서 세포 독성 T 림프구 확장이 대상 사이토 메갈로 바이러스, 엡스타인 - 바 바이러스, 그리고 아데노 바이러스

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

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 of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

탯줄 혈액에서 전구체 B-세포 하위 집합의 절연

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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

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

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

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

분리 및 나이브 CD4 T 림프구의 TH17 분화

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

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Nucleofection에 의해 인간의 THP-1 대 식세포의 고효율 형질

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

정렬 된 불멸화 된 B 세포로부터 재조합 인간 IgG 단일 클론 항체의 생성

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

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

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

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

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.