우리는 박사에 감사드립니다. 시약을 제공 마르쿠스 만츠와 어빙 와이즈맨. 우리는 또한 각각 의학 및 NTU 병원의 국립 대만 대학에서 유세포 분석 및 우선의 셀 정렬 핵심 시설과 두 번째 핵심 연구소가 제공하는 서비스를 인정합니다. 이 작품은 과학 기술, 대만의 교육부에 의해 지원되었다 (가장 102-2320-B-002-030-MY3)과 국민 건강 연구소, 대만 (NHRI-EX102-10256SI과 NHRI-EX103-10256SI).

C57BL / 6 야생형 마우스 국립 실험 동물 센터 (NLAC), 대만에서 구입 하였다. 모든 마우스는 사육 및 특정 병원균이없는 상태에서 유지되었다. 프로토콜 및 동물 사용 절차를 검토하고 의학의 국립 대만 대학의 기관 동물 관리 및 사용위원회 (허가 번호 : 20120075)에 의해 승인되었다. 또한, 연구진은 실험을 수행하는 동안 동물에 고통, 고통, 또는 고통의 가능성을 줄이기 위해 모든 노력을했다. 장갑을 착용하는 동안 설명하는 모든 절차는 실온에서 수행되었다. AC-6 피더 세포의 제조 (1) 주 : AC-6.21 (AC-6 짧은) (I. 와이즈맨, 스탠포드 대학에서 제공) 기질 세포 라인 (18)하는 것은 RPMI에서 유지되어야는 15 % 열 - 불 활성화 된 소 태아 혈청 (FBS)으로 보충. 피더 세포의 역할을하기 위해, AC-6 세포-γ 조사 3,000 방사선 (30Gy) 자신의 확산을 방지하기 위해 공동 문화 전에 일일와 있습니다. 참고 일그들의 통로 번호가 20 인 경우에 AC-6 세포는 유지 보수시 혼잡 방치하면 자신의 분화 지원 능력을 상실하는 경향이있다, 또는. <ol><li> 워시 AC-6 세포는 한 번에 1 ml의 둘 베코 인산염 완충 식염수 (DPBS)와 열망에 의해 DPBS를 제거합니다. AC-6 37 O C 3-5 분 동안 0.8 ml의 트립신 용액 (0.05 % 트립신 및 DPBS 0.5 mM의 EDTA), 그 다음 5 % CO 2와 함께 세포 배양 배지의 10 볼륨으로 희석하여 반응을 정지 치료 단일 세포 현탁액을합니다. </li><li> 5.9x10에서 종자 AC-6 세포는 4 세포 / 웰로 12 웰 플레이트와 5 %, 37 ℃로 배양 CO 2 O / N. 주 : AC-106 세포의 밀도가 CDC 생성을 선호한다 저밀도 매우 중요하다. 5.9x10의 밀도로 시드 아니라 AC-6 CLP를에서 올라가고의 유도를위한 최적 다음날에 의해 포화 상태에 도달 할 수 / 4 셀. </li><li> γ-조사 장치를 이용하여 3000 RAD (30 Gy의)에서 AC-6 세포를 조사. 노트: AC-6을 조사하는 데 사용되는 투여 량에서 세포 증식을 방지하고, 또 CLP를 행 수지상 분화에 필요한 사이토 카인과 세포 - 세포 접촉을 제공하기에 충분히 오래 생존 그들을 유지하기 위해 최적화된다. </li><li> 대기음 매체 (10 % 열 - 불 활성화 FBS, 50 μg의 / ㎖ 겐타 마이신과 50 μM의 β-머 캅토 에탄올로 RPMI) 완전한 RPMI 1 ㎖로 대체합니다. </li></ol> 마우스 2. 격리 BM 세포 <ol><li> CO 2 질식 자궁 전위에 의해 쥐를 희생. 해부 트레이에 마우스를 놓고 70 % 에탄올로 소독. 중반 복부에 절개를하고 하반신을 덮고있는 피부를 포함하여 마우스의 말단 부분에서 피부를 제거합니다. </li><li> 반 멸균 후드에서 쥐를 해부하다. 대퇴골과 경골을 해제하려면, 무릎 관절 아래 위의 대퇴골과 경골 클립. 가위를 사용하여 뼈에서 근육을 분리하고 RPMI을 완료 5 ㎖를 포함하는 6cm 페트리 접시에 뼈를 배치합니다. </li><li> 멸균 문화 후드로 이동합니다. 완전한 RPMI와 27G 바늘에 연결된 3 ML의 주사기를 채우십시오. 가위를 사용하여 뼈의 양쪽 끝을 잘라, 부드럽게 각각의 끝에서 완료되면 RPMI를 주입하여 뼈에서 골수를 플러시 27G 바늘을 삽입합니다. </li><li> 세포 상하 3-5 배 바늘없는 주사기를 사용하여 배양 접시에서 부드러운 피펫 팅에 의해 단일 ​​세포 현탁액을 제조. </li><li> 500 XG에서 5 분 동안 세포를 원심 분리기와 뜨는을 가만히 따르다. </li><li> 1 mL의 ACK 완충액 (150 mM의 CL NH 4, 10 mM의 KHCO 3, 0.1 mM의 EDTA) 1 분 동안 배양하고 완전 RPMI 10 부피로 희석하여 반응을 정지와를 Lyse 적혈구. 세포가 중력에 의해 죽은 세포 덩어리 및 조직 파편을 펠렛하기 위해 5 분간 방치한다. 참고 :이 때문에 가능한 세포의 정착에 세포가 손실 될 수로 이상 5 분을 서 있지 마십시오. </li><li> 새로운 튜브 떠나 파편에 천천히 캔트 세포 함유 뜨는원래 튜브 뒤에. </li><li> 다음, 세포 펠렛 제거하고 상층 액을 버린다 500 XG에서 5 분 동안 원심 분리기. </li><li> 총 BM 세포를 재현 탁 FACS 완충액 (1X PBS + 2 % FBS +를 1mM EDTA) 100 μL (전형적으로 4-6 X 107 BM 세포 한 마우스로부터 획득된다). </li></ol> 3. FACS 분석 및 CLP를의 정렬 <ol><li> 된 Fc 수용체를 차단하기 위해 1 ~ 2 분 동안 항 CD16 / 32 (하이 브리 도마 상층 액 10 클론 2.4G2, 50 μL / 반응 또는 1-2 μg의 / 반응) FACS 버퍼에 세포에 (단계 2.9)를 추가합니다. 주 : 안티 CD16 / 32 또한 과립구, 단핵구, 및 B 림프구를 비롯한 된 Fc 수용체를 발현하는 세포에 대한 항체의 비특이적 결합을 방지하기 위해 첨가 된 Fc 블록이라고 부른다. </li><li> 동시에 주변 광을 피하고, 얼음에 15 분 동안 (4 × 7 셀) 다음과 같은 형광 염료 표지 항체와 세포를 얼룩. 항체 : PE - 복합 혈통 마커 항 CD3 포함 (0.2 μg의 각) (17A2) 항 CD8 (53에서 6.7 사이), 항 B220 (RA3-6B2), 항 CD19 (eBio1D3), 항 -CD11b (M1 / 70), 안티 GR-1 (RB6-8C5), 안티 Thy1.1 (HIS51), 안티 NK1.1 (PK136), 안티 TER119 (TER-119), 및 안티 MHC-II (NIMR-4), 0.2 μg의 항 - C-KIT-를 PerCP-Cy5.5 (2B8), 1 μg의 안티 무서-1-FITC (D7), 0.4 μg의 안티 - M-CSFR-APC (AFS98), 0.2 μg의 항 IL-7Rα-PE-Cy7 (A7R34). </li><li> 500 X g에서 5 분 동안 3 ML의 외과 버퍼와 세포 및 원심 분리기를 씻으십시오. </li><li> 300 ㎕의 FACS에 세포가 40 μM 셀 스트레이너 버퍼 및 필터를 통해 세포를 재현 탁. </li><li> 남아있는 세포를 복구하고 동일한 셀 스트레이너를 사용하여 튜브를 정렬 멸균 외과로 필터링 추가 100 μL FACS 완충액으로 튜브를 씻으십시오. </li><li> 신호 검출에 대해 적절한 필터 및 전압을 사용하여 염색 후 즉시 유세포 분석을 수행한다. PE- 및 PE-Cy7-labelded 항체 및 633 nm의 라스를 검출하기 위해 561 nm의 레이저, FITC-, Cy5.5를 PerCP - 표지 된 항체를 검출하기 위해 488 nm의 레이저를 사용하여ER은 APC 표지 항체를 검출한다. </li><li> 씨 - 킷트 INT 무서 INT-1의 M-CSFR - - + IL-7Rα (단계 3.2에서 염색 등) 셀 소터를 사용하여 정렬 밖으로는 CLP를 다음 마커에 따른 LIN. 쿠션 완전 RPMI 8ml의 함유 15ml를 튜브에 세포를 수집 정렬. </li><li> 런의 ​​끝에서 셀 소터와 같이 분류 세포의 절대 수를 기록한다. 통상적으로, 5 × 4 CLP를 한 마우스로부터 BM을 얻을 수있다. </li></ol> 4. 공동 배양 CLP를 및 AC-6 <ol><li> 500 XG에서 10 분 동안 단계 3.7에서 분류 세포를 원심 분리하고 상층 액을 5 × 104 세포 / ㎖의 주위 세포 밀도를 얻기 위해 충분히 완전한 RPMI과 세포 펠렛을 재현 탁. 씨앗 500 세포 / 웰로 단계 1.4에서 준비 피더 세포를 포함하는 12 웰 플레이트. </li><li> 추가 100 NG FL 19 ㎖ / (후-Flt3L-IG는 M. 만츠, Universi 의해 제공 발현 시스템을 이용하여 내부에서 생성 된타이 병원 취리히, 스위스), 37 O C, 현미경 DC 개발의주기적인 영상 감시와 5 % CO 2에서 품어. 참고 : 시판되는 재조합 인간 또는 마우스 FL FL은 DC의 개발을 지원하기 위해 후 - Flt3L-IG 대용으로 사용될 수있다. </li><li> 12 일에 뜨는에서 세포를 수집하고 결과 상층 액을 결합 RPMI 배지를 완료 ㎖로 0.5 회 우물을 씻는다. 셀 스크레이퍼로 부착 세포를 0.5 ml의에게 신선한 매체를 추가하고 스크랩. </li><li> 500 x g에서 5 분 동안 세포 함유 배지에서 두 부분을 결합하고 원심. </li><li> 50 μL FACS 버퍼에 가만히 따르다 뜨는에 resuspend 세포. 50 μL 항 CD16 / 32 하이 브리 도마 뜨는 추가 된 Fc 수용체를 차단하는 1-2 분 동안 품어. </li><li> 열거하고 다음 항체 (0.05 μg의 각) 항 중 CD11c-APC (N418), 항 -CD11b-FITC 및 항 B220-PE 모든 세포를 염색. </li><li> 설명으로 세척하고 원심 분리기 세포단계 3.3에서 D. </li><li> 중 CD11c의 + 100 μL FACS 버퍼 게이트에서 세포를 재현 탁하고 CDCS (중 CD11c + CD11b를 + B220 -)에 대해 분석하고 올라가고 (중 CD11c + CD11b를 - B220의 +) (17). </li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+type+I+IFN-Flt3+ligand+axis+augments+plasmacytoid+dendritic+cell+development+from+common+lymphoid+progenitors.">A type I IFN-Flt3 ligand axis augments plasmacytoid dendritic cell development from common lymphoid progenitors.</a> J. Exp. Med. 210, 2515-2522 (2013).</li><li>Whitlock, C. A., Tidmarsh, G. F., Muller-Sieburg, C., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cell+lines+with+lymphopoietic+activity+express+high+levels+of+a+pre-B+neoplasia-associated+molecule.">Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule.</a> Cell. 48, 1009-1021 (1987).</li><li>Onai, N., Obata-Onai, A., Tussiwand, R., Lanzavecchia, A., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Flt3+signal+transduction+cascade+rescues+and+enhances+type+I+interferon-producing+and+dendritic+cell+development.">Activation of the Flt3 signal transduction cascade rescues and enhances type I interferon-producing and dendritic cell development.</a> J. Exp. Med. 203, 227-238 (2006).</li><li>Szilvassy, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia+inhibitory+factor+upregulates+cytokine+expression+by+a+murine+stromal+cell+line+enabling+the+maintenance+of+highly+enriched+competitive+repopulating+stem+cells.">Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.</a> Blood. 87, 4618-4628 (1996).</li><li>Arcanjo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+heparan+sulfate+derived+from+murine+hemopoietic+stromal+cell+lines:+a+bone+marrow-derived+cell+line+S17+and+a+fetal+liver-derived+cell+line+AFT024.">Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024.</a> J. Cell. Biochem. 87, 160-172 (2002).</li><li>Onai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+progenitor+with+prominent+plasmacytoid+dendritic+cell+developmental+potential.">A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential.</a> Immunity. 38, 943-957 (2013).</li><li>Whitlock, C. A., Muller-Sieburg, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+B-lymphoid+cultures+from+murine+bone+marrow+establishment+and+cloning+by+using+stromal+cell+line+AC+6.21.">Long-term B-lymphoid cultures from murine bone marrow establishment and cloning by using stromal cell line AC 6.21.</a> Methods Mol. Biol. 75, 231-248 (1997).</li><li>Chen, Y. -. L., Chang, S., Chen, T. -. T., Lee, C. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Generation+of+Plasmacytoid+Dendritic+Cell+from+Common+Lymphoid+Progenitors+by+Flt3+Ligand.">Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand.</a> PLoS ONE. 10 (8), e0135217 (2015).</li></ol>

Authors have nothing to disclose.

여기에서 우리는 CLP를 소수의에서, 특히 체외 배양 수지상의 강력한 생성을위한 시스템 및 올라가고을 설명합니다. 이 배양 시스템의 고유성은 피더로서 AC-106 세포, 간질 세포주의 이용에 기인한다. 이 방법은 예컨대 IL-7, SCF, M-CSF 및 FL 20뿐만 아니라 사이토 카인을 제공하기 위해 도시 한 내용뿐만 아니라, 필요한 세포 간 접촉 (21)은 DC 개발을 지원한다. AC-106 세포는 여러 ?...

수지상 세포 (DCS)는 면역 반응 1을 제어하는데 중요한 역할을 전문 항원 제시 세포이다. DC가 이종 있지만, 그들이 대체로 두 집단으로 분류 될 수 있고, 종래의 DC (CDCS) 및 형질 수지상 (PDCS) 2,3-. 림프 기관 외에 CDCS 및 PDCS 또한 폐, 소장, 피부 (2)를 포함한 조직에서 발견된다. CDCS 및 올라가고의 형태는 수상 돌기 모양의 돌기보다 형질 세포가 같은 것을 올라가고의 모양을 나타내는 CDCS으로 다르​​다. 또한, 일반적인 마우스 DC 마커, CD11c 양성은 더 높게 올라가고보다 CDCS에 표현된다. 올라가고 2보다 MHC 클래스 II의 높은 수준과 보조 자극 분자를 표현 둘 CD24 + CDCS, - CDCS와 CD11b를 - 또한, CDCS는 더는 CD11b + CD24로 나눌 수 있습니다. 성숙한 PDCS는 반면에, 선택적 Siglec-H 및 BST2 4를 발현하는 것으로 나타났다. 에프unctionally, CDCS는 올라가고보다 제시 세포보다 항원은; 그러나 올라가고 내가 바이러스 감염이나 염증 자극 5시 인터페론 유형의 광대 한 양을 생성 할 수 있습니다. 모두 CDCS 및 올라가고는 짧은 수명이며, 따라서 지속적으로 골수 (BM) 6,7 내 조상에서 보충해야합니다. 치명적-조사 된 마우스에 공통 골수 전구 세포 (CMP들) 및 일반 림프 조상 (CLP를)의 입양 전송 CDCS과 올라가고 양쪽 계통 8-10에서 생성 될 수 있음을 보여줍니다. 그러나, RAG1 / 2를 표현하고 고등학교의 궤적 (11, 12)에서 재 배열 DJ 세그먼트를 가지고 올라가고의 부분 집합이있다. 이 세포는 B220 마커의 발현, 핵산 감지 수용체 (TLR7 / TLR9) 및 전사 인자 (Spib 및 Bcl11a) (13)를 포함하는 B 림프구와 분자 유사성을 공유하고, 림프 혈통의 서명 될 것으로 모두가 있습니다. 따라서, CLPS 때문에 계보 유사성 올라가고의 체외 발생에 대한 좋은 선택 일 수있다. 인간 및 마우스 모두 CDCS 및 PDCS의 빈도가 매우 낮은 반면 6, 수지상 세포, 특히 CDCS는, (예를 GM-CSF 11,14 또는 FLT3 리간드와 같은 사이토 카인의 존재 BM 또는 전구 세포로부터 in vitro에서 생성 될 수있는 FL ) 공급 장치가없는 시스템 11,15,16를 사용하여. 불행하게도, 그러나, FL 11,15,16을 사용하여 시험 관내에서 PDC의 다수를 생성하는 것은 불가능하다. 이전에, 우리는 PDCS 효율적 AC-6 급전 시스템 (17)을 사용하여 시험 관내에서 CLP를 생성 할 수 있다는 것을 보여 주었다. 배양 시스템에서 AC-6 간질 세포주를 사용하는 장점은 세포 - 세포 접촉 CLP를 PDCS로부터 많은 수의 생성을 지원하는 사이토 카인의 분비를 제공한다는 것이다. 이 시스템으로 생산이 매우 강력하지만, 아래에 설명 된 절차에주의 복제 내가 필요합니다N 순서는 올라가고의 효율적 생성을 보장합니다.

<table><tbody><tr><td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td><td>Biolegend</td><td>108408</td><td>linage marker</td></tr><tr><td>Anti-mouse NK1.1 (PE), clone PK136</td><td>Biolegend</td><td>108708</td><td>linage marker</td></tr><tr><td>Anti-mouse&amp;nbsp; CD11b (PE), cloneM1/70</td><td>Biolegend</td><td>101208</td><td>linage marker</td></tr><tr><td>Anti-mouse CD19 (PE), clone eBio1D3</td><td>Biolegend</td><td>115508</td><td>linage marker</td></tr><tr><td>Anti-mouse B220 (PE), clone RA3-6B2</td><td>Biolegend</td><td>103208</td><td>linage marker/FACS</td></tr><tr><td>Anti-mouse CD3 (PE), clone 17A2</td><td>Biolegend</td><td>100308</td><td>linage marker</td></tr><tr><td>Anti-mouse CD8a (PE), clone 53-6.7</td><td>Biolegend</td><td>100707</td><td>linage marker</td></tr><tr><td>Anti-mouse MHC-II (PE), clone NIMR-4</td><td>Biolegend</td><td>107608</td><td>linage marker</td></tr><tr><td>Anti-mouse Ter119 (PE), clone TER-119</td><td>Biolegend</td><td>116208</td><td>linage marker</td></tr><tr><td>Anti-mouse Thy1.1 (PE), clone HIS51</td><td>eBioscience</td><td>12-0900-83</td><td>linage marker</td></tr><tr><td>Anti-mouse M-CSFR (APC), clone AFS98</td><td>Biolegend</td><td>135510</td><td>FACS</td></tr><tr><td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td><td>Biolegend</td><td>105824</td><td>FACS</td></tr><tr><td>Anti-mouse Sca-1 (FITC), clone D7</td><td>Biolegend</td><td>108106</td><td>FACS</td></tr><tr><td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td><td>Biolegend</td><td>135014</td><td>FACS</td></tr><tr><td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td><td>Biolegend</td><td>117328</td><td>FACS</td></tr><tr><td>Anti-mouse CD11b (FITC), clone M1/70</td><td>Biolegend</td><td>101206</td><td>FACS</td></tr><tr><td>FACSAria III</td><td>BD Biosciences</td><td></td><td>Cell sorter</td></tr><tr><td>FACS sorting tube&amp;nbsp;</td><td>BD Biosciences</td><td>352054</td><td></td></tr><tr><td>FlowJo</td><td>FlowJo LLC</td><td></td><td>Flow analysis sofrware</td></tr></tbody></table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

형질 수지상 세포 (올라가고은) 내가 감염 또는 염증 반응 동안 응답 활성화 (IFN-1) 생산 세포를 인터페론 강력한 유형입니다. 불행하게도, PDC 기능의 연구는 림프 기관에 낮은 주파수에 의해 방해하고, 체외 DC 생성을위한 기존의 방법은 주로 올라가고 이상 CDCS의 생산을 선호. 여기에 우리가 효율적으로 체외에서 일반적인 림프 전구 세포 (CLP를)에서 올라가고을 생성하는 독특한 접근 방식을 제시한다. 특히, 프로토콜은 골수로부터 CLP를 정화 및 FLT3 리간드의 존재하에 γ 조사 된 AC-6 피더 세포와 공동 배양함으로써 PDCS를 생성하는 방법을 자세히 설명했다. 이 문화 시스템의 고유 한 특징은 CLP를가 AC-6 세포 아래 마이그레이션 및 조약돌 영역 형성 세포, PDC의 확장을위한 중요한 단계가 될 것입니다. 형태 학적으로 별개의 수지상, 즉 올라가고 및 CDCS는 7 조성 후 약 2 주 생성 된최적 조건 하에서 0-90%의 PDCS. 일반적으로이 방법에 의해 생성 PDCS의 수는 대략 CLP를 시딩 수의 100 배이다. 따라서, 이것은 견고 이들 세포의 발달 및 기능에 추가 연구를 용이하게하기 위해 필요한 PDCS의 다수를 생성되는 신규 한 시스템이다.

4-6x10 7 BM 세포의 총 일반적으로 대퇴골 한 6 ~ 8 주령, 야생형 C57BL / 6 마우스의 경골에서 격리됩니다. CLP를 밖으로 정렬하려면, 총 BM 세포는 혈통 마커 (CD3, CD8, B220, CD19, CD11b를, GR-1, Thy1.1, NK1.1, TER119 및 MHC-II), 안티에 대한 PE - 복합 항체로 염색 -c-KIT-를 PerCP / Cy5.5, 안티 무서-1-FITC, 안티 - M-CSFR-APC 및 항 IL-7Rα-PE / Cy7는 분석하고 세포 분류기로 분류. CLP에 대한 선별 전략은 그림 1에...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

AC-6 공급 시스템을 사용하여 일반 림프 전구 세포에서 쥐 형질 수지상 세포의 체외 세대

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

in-vitro-generation-murine-plasmacytoid-dendritic-cells-from-common

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.4K 조회수.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

비디오: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

골수 엽 성의 전구 물질에서 골수양 돌기 세포의 문화

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

생물학

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

골수의 생성은 2 광자 영상에서 사용하기 위해 Murine 돌기 세포를 유래

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

조혈 전구 세포의 입양 전송을 사용 Peyer의 패치에 쥐과 Plasmacytoid 수지상 세포의 개발을 평가

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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 ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

대사 표현형을 각기 다른 미성숙, 성숙 및 Tolerogenic 수지상 세포의 생성

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

골수성 창시자 및 정렬 전략 소설 셀을 사용 하 여 Murine 골에서 수지상 세포 선구자의 큰 숫자의 생성

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

개발 및 Murine Tolerogenic 수지상 세포의 기능적 특성

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

의학

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

In vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors(일반 림프구 전구 세포에서 Plasmacytoid 수지상 세포의 체외 생성)

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...