1. PBMC의 분리 <ol><li> BD Vacutainer CPT 셀 준비 튜브 나트륨 헤파린과에 혈액 (7.5-8.0 ML)를 그립니다. 튜브는 나트륨 헤파린의 항응고제와 Ficoll - Hypaque 밀도 유체, 더하기 두 액체를 분리 폴리에스터 겔 장벽을 포함하고 있습니다. 1,800 X g에서 20 분 수평 로터 (스윙 아웃 헤드)에 실온 (18 ° C 25 ° C)에서 튜브 / 혈액 샘플을 원심 분리기. 브레이크를 원심 분리기 전환합니다. </li><li> 원심 분리 후, 레이어의 순서 (위에서 아래에서 본) 다음과 같이 발생합니다 :) 플라즈마 - B) 말초 혈액 mononuclear 세포 (PBMC)과 혈소판 - C) 밀도 솔루션 - D) 폴리에스터 겔 - E) granulocytes - F) 적혈구 (그림 1). </li><li> 셀 계층을 방해하지 않고 계면 위의 플라즈마의 5~10mm를 떠나, 플라즈마 층의 일부를 수집합니다. 플라즈마는 문화 (섹션 2.5)을 위해 사용될 수 있습니다. </li><li> 15 ML 원뿔 관에 피펫 및 이전과 계면에서 농축 분율 (PBMC)를 수확. </li><li> 10 ML과 PBMC을 씻 는다 인산염 버퍼 반전 튜브에 의해 생리 (PBS), 5 번, 그리고 400 X g에서 5 분 원심 분리기. </li><li> 두 번 세척 단계를 반복한 다음 PBS 5 ML에있는 세포 펠렛을 resuspend. 휴대폰 번호를 확인합니다. 보통 1.3 X10 6 셀은 전체 혈액의 1 ML에서 회복됩니다. </li><li> 주파수 및 유동세포계측법 (섹션 3) (그림 2)에 의해 PBMC에서 γδ T 세포의 표현형를 결정합니다. </li></ol> 2. γδ ​​T 세포의 확장 <ol><li> 상온에서 400 X g에서 5 분 15 ML 원뿔 튜브에 세포를 원심 분리기 suspensions하고 supernatants 폐기하십시오. </li><li> 각각 1000 IU / ML 5 μm의 최종 농도에 인간 IL - 2 (IL - 2)와 zoledronate (Zometa)를 추가하여 문화 매체 (CM)를 준비합니다. ALyS203 (셀 과학 기술 연구소) 또는 OpTmizer (Invitrogen) 미디어 γδ T 세포 (참조 6 9 자세한 정보)의 좋은 확장을 지원합니다. Zometa는 액체 형태 (4 mg/5-ml의 약병)에 제공됩니다. 5 μm의 솔루션을 준비하려면, 문화 매체의 30 ML에 Zometa 50 μl를 추가합니다. </li><li> Resuspend 문화 매체 세포 펠릿 및 1x10 6 세포 / ML을 조절할 수 있습니다. </li><li> 24 - 잘 플레이트의 각 우물에 1x10 6 세포를 포함하는 CM의 Pipet 1 ML. 대규모 문화, 전지 판 우물, 요리,이나 플라스크의 표면 영역에 따라이 0.5 X 10 6 세포 / cm에서 씨앗을하실 수 있습니다. </li><li> 그것은 문화의 볼륨 (24 잘 플레이트의 각 물론 100 μl)의 약 10 % 있도록 autologous 플라즈마 (섹션 1.3), 풀링 인간 AB 세라, 또는 FCS를 추가합니다. humidified 37 접시를 놓고 ° C 5 % 24-48 시간에 대한 CO 2 배양기. </li><li> 0.5-2 X 10 6 세포 / ML의 세포 밀도의 문화를 유지합니다. 신선한 매체마다 2-3일 인간 IL - 2 (1000 IU / ML) 전용 (Zometa없이)이 포함된 추가하고, 세포 증식의 정도 (그림 3)에 의하면, 새로운 우물하거나 필요한만큼 flasks으로 교양 세포를 전송합니다. 공급 플라즈마 또는 혈청 농도가 적어도 1% 유지 수 있도록 매체 혈청. </li><li> 일 12-14과 주파수, 표현형 및 유동세포계측법에 의해 γδ T 세포 (아래 참조)의 기능을 결정에 수확 세포. </li></ol> 3. 유동세포계측법에 의해 Phenotypic 분석 <ol><li> 형광 - 활성화된 셀 정렬 (외과) 튜브 2 X 10 5 세포를 포함하는 200 μl 샘플을 전송합니다. </li><li> 차가운 PBS 2 ML 추가 400 X g에서 5 분 원심 분리기. 그런 다음, 50 μl 외과 버퍼 (PBS + 1% FCS + 0.1 %의 나트륨 azide)에 알약을 resuspend. 샘플 (단클론 항체가 표 1에 나와 있습니다) 각 항체의 5 μl를 추가합니다. </li><li> 20 분 동안 짙은 어둠 속에서 얼음 품어. </li><li> 각 샘플 2 ML의 외과 버퍼를 추가한 다음 소용돌이. 4 400 X g ° C.에서 5 분 원심 분리기 샘플 조심스럽게 뜨는을 가만히 따르다. </li><li> 300 μl 외과 버퍼와 와동에있는 세포를 Resuspend. 흐름 cytometer (그림 2)에서 샘플을 분석할 수 있습니다. </li></ol> 4. IFN - γ 생산 분석 10 <ol><li> 분석 전날, RPMI 1640 미디어 플러스 Zometa (5 μm의) (이하 Z - Daudi 지정)과 하룻밤 10% FCS (RPMI - 10)에 culturing 3-5 X10 5 Daudi 세포 / ML로 자극기 전지를 준비합니다. </li><li> 2x10 6 셀 / ML에서 RPMI - 10 Z - Daudi 및 resuspend를 수집합니다. 둥근 바닥 96 - 웰 플레이트의 각 잘하는 Z - Daudi (2x10 5) 100 μl를 추가합니다. </li><li> RPMI - 10은 20 μg / ML에서 Brefeldin를 포함에 2x10 6 셀 / ML에서 γδ T 세포를 준비합니다. 각 잘 포함 Z - Daudi 전지에 γδ T 세포 현탁액 (2x10 5) 100 μl를 전송하거나 우물 (100 단 RPMI - 10 μl, 또는 phorbol 20 NG / ML 12 - myristate 13 - 아세테이트와 RPMI - 10 제어[PMA] 플러스 2 μg / ionomycin의 ML). </li><li> 몇 시간을 아래와 pipetting하여 섞는다. 37 ° C 5 % CO 2 배양기에서 4 시간을위한 부화. </li><li> 4 ° C에서 400 X g에서 5 분 접시를 원심 차가운 PBS 200 μl의 알약을 resuspend. </li><li> 외과 튜브에 샘플을 전송합니다. 차가운 PBS 4 ML을 추가하고 4 400 X g에서 5 분 튜브를 원심 ° C. </li><li> FITC - 복합 백신 TCRVγ9 (5 μl)와 PE/Cy5-conjugated 안티 인 경우에는 3 번 CD mAb (2.5 μl)와 외과 버퍼의 50 μl의 알약을 Resuspend. 부화는 실온에서 15 분 빛으로부터 보호. </li><li> IntraPrep 시약 1 100 μl를 추가하고 실온에서 15 분 알을 품다. 상온에서 400 X g에서 5 분 각 튜브와 원심 분리기에 PBS 4 ML을 추가합니다. </li><li> 흡인에 의해 표면에 뜨는를 제거하고 IntraPrep 시약 2 100 μl를 추가합니다. 떨지 않고 상온에서 5 분 알을 품다. </li><li> 테스트 튜브에 PE - 복합 백신 IFN - γ mAb 5 μl를 추가합니다. 부화는 실온에서 15 분 빛으로부터 보호. </li><li> 상온에서 400 X g에서 5 분 각 튜브와 원심 분리기에 PBS 4 ML을 추가합니다. 흡인에 의해 표면에 뜨는를 제거하고 외과 버퍼의 0.5 ML에있는 세포 펠렛을 resuspend. </li><li> 흐름 cytometer에 의한 세포를 분석할 수 있습니다. 인 경우에는 3 번 CD + TCRVγ9 + 세포에서 IFN 게이트 - γ (그림 4)의 표현을 확인합니다. </li></ol> 5. 대표 결과 : 문화의 개시에 PBMC에서 γδ T 세포의 비율을 결정하는 것이 중요합니다. 마찬가지로 그림에 표시됩니다. 2 A, 인 경우에는 3 번 CD + TCRVγ9의 비율은 + PBMC에서 γδ T 세포는 하루에 0에 1.6 %되었습니다. 중앙 메모리 phenotypes - 지배 집단은 CD27 + CD45RA + 순진한 또는 CD27 + CD45RA했다. γδ T 세포가 효율적으로 자극되었을 때, 그들은 (그림 3 A와 B) 일 3-5에 클러스터를 형성. 클러스터 형성이 지연되었을 때, 같은 다른 세포 유형의 성장 CD4 + 또는 CD8 + αβ T 세포 또는 NK 세포가 γδ T 세포 (그림 3 C 및 D)의 성장을 지배 수 있습니다. 문화 날로부터 14 일 후, γδ T 세포의 빈도가 성공적으로 γδ T 세포 배양 (그림 2 E)에서 교양 세포의 이상이 93.8 %로 증가했다. 교양 γδ T 세포 upregulated NKG2D 및 CD69 표현 (그림 2 G 및 H). 그들은 CD27 표시 - CD45RA - 효과기 메모리 표현형 (그림 2 F). γδ ​​T 세포의 기능이 시토킨 생산 및 세포 독성에 관한 평가했다. 세포내 IFN - γ 얼룩 γδ T 세포는 PMA / ionomycin 치료 또는 Z - Daudi 세포에 대한 응답으로 IFN - γ를 생산 것을 입증되는 zoledronate 치료 후 누적된 IPP (그림 4). 이러한 결과는 zoledronate 효율적으로 자극하고 기능 γδ T 세포를 확장할 수 있음을 나타냅니다. <table border="1"><tbody><tr><td> 튜브 </td><td> FITC </td><td> PE </td><td> ECD </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> 인 경우에는 3 번 CD에 들어 </td><td> CD19 </td><td> CD45 </td><td> CD14 </td></tr><tr><td> 2 </td><td> 인 경우에는 3 번 CD에 들어 </td><td> TCRαβ </td><td> CD4 </td><td> CD8 </td></tr><tr><td> 3 </td><td> 인 경우에는 3 번 CD에 들어 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCR Vγ9 </td><td> TCRαβ </td><td> CD45 </td><td> 인 경우에는 3 번 CD에 들어 </td></tr><tr><td> 5 </td><td> TCR Vγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCR Vγ9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCR Vγ9 </td><td> 마우스 IgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCR Vγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCR Vγ9 </td><td></td><td> 마우스 IgG1 </td><td> 마우스 IgG1 </td></tr></tbody></table> 표 1. γδ T 세포의 여러 가지 빛깔의의 얼룩에 사용되는 단클론 항체. 우리 연구실에서 수행 γδ T 세포의 phenotypic 분석 예제는 그림에 표시됩니다. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="그림 1"/> 그림 1. PBMC의 분리. 블러드 (7.5-8.0 ML)은 BD Vacutainer CPT 셀 준비 튜브 나트륨 헤파린과에 직접 그려 1,800 X g에서 20 분 centrifuged입니다. 원심 분리 후, 같은 결과 레이어는 처음부터 보로 볼 수ttom :) 플라즈마 - B) PBMC 및 혈소판 - C) 밀도 솔루션 - D) 폴리에스터 겔 - E) Granulocytes - F) 적혈구. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="그림 2"/> 그림 2. γδ T 세포의 일반적인 표면 표현형. PBMC는 14 일 동안 zoledronate 및 IL - 2와 함께 자극했다. 세포와 스테인드되었습니다 FITC - 라벨 안티 TCR γδ T Vγ9 및 세포 (A와 E)의 확장을 모니터하는 안티 - 인 경우에는 3 번 CD에 들어 PE/Cy5-labeled. γδ ​​T 세포는 TCRVγ9 자신의 표현으로 식별되었고, CD27 및 CD45RA (B와 F), NKG2D (C와 G), 또는 CD69 (D 및 H) 자신의 표현을 조사했다. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="그림 3"/> 그림 3. 대표 γδ T 세포 문화. PBMC는 IL - 2 (1000 IU / ML) 및 zoledronate (5 μm의)와 자극했다. 대표 필드 (IX71 거꾸로 현미경 [올림푸스] X 200) 표시됩니다. γδ ​​T 세포의 클러스터 및 집계는 γδ T 세포가 성공적으로 확장되었습니다 3 일 (A)과 5 일 (B)에 볼 수 있습니다. γδ ​​T 세포 성장 (C 및 D) 충분하지 때 대조적으로, 어떤 클러스터 또는 집계는 관찰되지 않았습니다. <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="그림 4"/> 그림 4. IFN - γ 생산. γδ ​​T 세포는 RPMI - 10 (A) 또는 PMA / ionomycin (B) 또는 4 시간에 대한 Z - Daudi (C)와 incubated했다. 첫째, TCRVγ9의 표면 표현은 스테인드했습니다 그리고 IFN - γ 생산 세포내 IFN - γ 염색법에 의해 조사되었다. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="그림 5"/> 그림 5. γδ T 세포 문화의 속도론. 교양 세포 (A) 절대 번호, (B) γδ T 세포, 그리고 지정된 시간 지점에서 γδ T 세포 (C) 절대 번호의 비율입니다.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T+cell+receptor+&#947;&#948;+cells+recognize+endogenous+mevalonate+metabolites+in+tumor+cells.">Human T cell receptor &#947;&#948; cells recognize endogenous mevalonate metabolites in tumor cells.</a> J Exp Med. 197, 163-168 (2003).</li><li>Kabelitz, D., Wesch, D., He, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+of+gammadelta+T+cells+in+tumor+immunology.">Perspectives of gammadelta T cells in tumor immunology.</a> Cancer Res. 67, 5-8 (2007).</li><li>Roelofs, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+monocytes+are+responsible+for+gammadelta+T+cell+activation+induced+by+zoledronic+acid+through+accumulation+of+IPP/DMAPP.">Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP.</a> Br J Haematol. 144, 245-250 (2009).</li><li>Dieli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+&#947;&#948;+T-lymphocyte+effector+functions+by+bisphosphonate+zoledronic+acid+in+cancer+patients+in+vivo.">Induction of &#947;&#948; T-lymphocyte effector functions by bisphosphonate zoledronic acid in cancer patients in vivo.</a> Blood. 102, 2310-2311 (2003).</li><li>Kondo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronate+facilitates+large-scale+ex+vivo+expansion+of+functional+&#947;&#948;+T+cells+from+cancer+patients+for+use+in+adoptive+immunotherapy.">Zoledronate facilitates large-scale ex vivo expansion of functional &#947;&#948; T cells from cancer patients for use in adoptive immunotherapy.</a> Cytotherapy. 10, 842-856 (2008).</li><li>Espinosa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+biological+activity+of+bromohydrin+pyrophosphate,+a+potent+stimulator+of+human+&#947;&#948;+T+cells.">Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human &#947;&#948; T cells.</a> J Biol Chem. 276, 18337-18344 (2001).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+profile+and+anti-tumor+effects+of+adoptive+immunotherapy+using+&#947;&#948;+T+cells+against+advanced+renal+cell+carcinoma:+a+pilot+study.">Safety profile and anti-tumor effects of adoptive immunotherapy using &#947;&#948; T cells against advanced renal cell carcinoma: a pilot study.</a> Cancer Immunol Immunother. 56, 469-476 (2007).</li><li>Murali-Krishna, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+antigen-specific+CD8+T+cells:+a+reevaluation+of+bystander+activation+during+viral+infection.">Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.</a> Immunity. 8, 177-187 (1998).</li><li>Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+culture+medium+on+the+expansion+of+T+cells+for+immunotherapy.">Impact of culture medium on the expansion of T cells for immunotherapy.</a> Cytotherapy. 11, 936-946 (2009).</li><li>Abe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+immunological+evaluation+of+zoledronate-activated+V&#947;9+&#947;&#948;T-cell-based+immunotherapy+for+patients+with+multiple+myeloma.">Clinical and immunological evaluation of zoledronate-activated V&#947;9 &#947;&#948;T-cell-based immunotherapy for patients with multiple myeloma.</a> Exp Hematol. 37, 956-968 (2009).</li><li>Nakajima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+study+of+adoptive+immunotherapy+for+recurrent+non-small-cell+lung+cancer+patients+with+autologous+&#947;&#948;+T+cells.">A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous &#947;&#948; T cells.</a> Eur J Cardiothorac Surg. 37, 1191-1197 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기서 제시하는 방법은 PBMC에서 γδ T 세포의 효율적인 확장이 가능합니다. γδ ​​T 세포가 활성화되고 확대 zoledronate로와 IL - 2는 시토킨 생산 및 세포 독성에 의해 반사 완료 효과기 기능을 개발할 수 있습니다. 그것은보고되었음을 합성 phosphoantigens의 bromohydrin 나트륨 (BrHPP) 및 2 - 메틸 - 3 - butenyl - 1 - 나트륨 (2M3B1PP)는 또한 γδ T 세포를 확장하고 있지만, 그들은 상업적으로 사용할 수 없습니다....

<table border="1"><tbody><tr><th> 시약 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> ZOMETA </td><td> 노바티스 제약 K. K </td><td></td><td> zoledronate </td></tr><tr><td> PROLEUKIN </td><td> 노바티스 제약 </td><td></td><td> 인간 재조합 IL - 2 </td></tr><tr><td> BD Vacutainer CPT 셀 준비 튜브 나트륨 헤파린과 함께 </td><td> BD </td><td> 362753 </td><td></td></tr><tr><td> RPMI1640 </td><td> Invitrogen </td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203 - 중간 </td><td> 셀 과학 기술 연구소 </td><td> 0301-7 </td><td></td></tr><tr><td> OpTmizer </td><td> Invitrogen </td><td> 0080022SA </td><td></td></tr><tr><td> brefeldin </td><td> 시그마 </td><td> B5936 - 200UL </td><td></td></tr><tr><td> phorbol 12 - myristate 13 - 아세테이트 (PMA) </td><td> 시그마 </td><td> P1585 - 1MG </td><td></td></tr><tr><td> ionomycin </td><td> 시그마 </td><td> 13909 - 1ML </td><td></td></tr><tr><td> IntraPrep </td><td> 베크맨 쿨터 </td><td> A07803 </td><td></td></tr><tr><td> 안티 - 인간 인 경우에는 3 번 CD - FITC 또는 PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> 안티 - 인간 CD4 - ECD </td><td> 베크맨 쿨터 </td><td> 6,604,727 </td><td></td></tr><tr><td> 안티 - 인간 CD8-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> 6,607,011 </td><td></td></tr><tr><td> 안티 - 인간 CD14-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07765 </td><td></td></tr><tr><td> 안티 - 인간 CD19 - PE </td><td> 베크맨 쿨터 </td><td> A07769 </td><td></td></tr><tr><td> 안티 - 인간 CD45 - ECD </td><td> 베크맨 쿨터 </td><td> A07784 </td><td></td></tr><tr><td> 안티 - 인간 CD56-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07789 </td><td></td></tr><tr><td> 안티 - 인간 TCRαβ - PE </td><td> 베크맨 쿨터 </td><td> A39499 </td><td></td></tr><tr><td> 안티 인간 TCR - FITC Vγ9 </td><td> 베크맨 쿨터 </td><td> IM1463 </td><td></td></tr><tr><td> 안티 - 인간 CD27-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> 6,607,107 </td><td></td></tr><tr><td> 안티 - 인간 CD45RA - ECD </td><td> 베크맨 쿨터 </td><td> IM2711 </td><td></td></tr><tr><td> 안티 - 인간 CD69 - PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td> 안티 - 인간 NKG2D - PE </td><td> 베크맨 쿨터 </td><td> A08934 </td><td></td></tr><tr><td> 안티 humal IFNγ - PE </td><td> 베크맨 쿨터 </td><td> IM2717U </td><td></td></tr><tr><td> 마우스 IgG1 isotype 제어 PE </td><td> 베크맨 쿨터 </td><td> A07796 </td><td></td></tr><tr><td> 마우스 IgG1 isotype 제어 ECD 또는 PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

인간 γδ T 세포가 인식하고 스트레스 유발 항원의 다양한 반응, 따라서 본래 광범위한 안티 종양 및 안티 infective 활동을 개발하고 있습니다. 말초혈에서 γδ T 세포의 1 대부분 Vγ9Vδ2 T 세포 수용체 있습니다. 이러한 세포의 주요 histocompatibility 복합 독립적인 방식으로 항원을 인식하고 강한 cytolytic과 Th1과 같은 이펙터 기능을 개발할 수 있습니다. 1 따라서, γδ T 세포가 암 immunotherapy에 대한 매력적인 후보 효과기 세포 수 있습니다. Vγ9Vδ2 T 세포 같은 phosphoantigens에 대응 (E) -4 - 히드록시 -3 - 메틸 -하지만 - 2 - enyl 나트륨 (HMBPP), isoprenoid 생합성 통해 박테리아에 합성하는, 생산 2 isopentenyl 나트륨 (IPP), mevalonate 경로 3. 생리적 조건을 통해 진핵 세포에서 nontransformed 셀에 IPP의 세대 γδ T 세포의 활성화를 위해 충분하지 않습니다. 종양 세포에서 mevalonate 경로의 Dysregulation는 IPP의 축적과 γδ T 세포의 활성화로 연결됩니다. 3 aminobisphosphonates (예 : pamidronate 또는 zoledronate 등) farnesyl 나트륨의 synthase (FPPS), mevalonate 경로에서 IPP의 하류를 연기 효소의 세포내 수준을 억제하기 때문에 IPP 및 γδ T 세포 인식에 sensitibity는 therapeutically aminobisphosphonates 증가 수 있습니다. IPP 축적 aminobisphosphonates와 γδ T 세포를 활성화하여 암 우리에게 immunotherapy 수 aminobisphosphonates의 pharmacologically 관련 농도와 종양 세포보다 nontransfomred 세포에 덜 효율적이다. PBMC는 aminobisphosphonates로 치료하는 경우 4 흥미롭게도, IPP 때문에 효율의 monocytes에 쌓입니다 이러한 세포로 약물 이해. IPP 항원 제시 세포되고 주변 혈액에 Vγ9Vδ2 T 세포를 자극 축적 5 Monocytes가 6. 이러한 메커니즘을 바탕으로, 우리는 zoledronate와 인터루킨를 사용하여 γδ T 세포 문화의 대규모 확장을위한 기술을 개발 -2 (IL - 2). γδ T 세포의 확장 7 다른 방법은 합성 phosphoantigens의 bromohydrin 나트륨 (BrHPP) 8 2 - 메틸 - 3 - butenyl - 1 - 나트륨 (2M3B1PP)를 활용 9. 이러한 방법 중 모든 전 수 입양 immunotherapy에 사용하기 위해 γδ T 세포의 다수의 결과로 생체내 확장. 단, zoledronate은 FDA의 승인을 상용 시약입니다. CD45RA - - Zoledronate - 확장 γδ T 세포는 CD27 표시 효과기 메모리 표현형과 그들의 기능은 IFN - γ 생산 분석 7 평가할 수 있습니다.

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  Î³Î´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

주변 혈액 mononuclear 세포 (PBMC)에서 γδ T 세포를 확장하는 방법을 설명합니다. PBMC - 파생 γδ T 세포는 자극과 zoledronate과 인터루킨 - 2 (IL - 2)를 사용 확장됩니다. γδ ​​T 세포의 대규모 확장이 암의 autologous 세포 immunotherapy에 적용할 수 있습니다.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

26.6K 조회수.  University of Tokyo Hospital. 주변 혈액 mononuclear 세포 (PBMC)에서 γδ T 세포를 확장하는 방법을 설명합니다. PBMC - 파생 γδ T 세포는 자극과 zoledronate과 인터루킨 - 2 (IL - 2)를 사용 확장됩니다. γδ ​​T 세포의 대규모 확장이 암의 autologous 세포 immunotherapy에 적용할 수 있습니다.

비디오: Expansion of Human Peripheral Blood  γδ T Cells using Zoledronate

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

연구

면역학 및 감염병학

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

임상 신청<em&gt; 잠자는 숲속의 미녀</em유전자 주변과 배꼽 탯줄 혈액에서 T 세포를 수정하는 셀을 제시&gt;과 인공 항원

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 감염 CD4의 타고난 감지 평가<sup&gt; +</sup&gt;를 사용하여 형질 수지상 세포에 의한 T 세포<em&gt; 예 생체 내</em&gt; 공동 문화 시스템.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

사람 말초 혈액의 Immunophenotypic 분석에 대한 항체 칵테일을 준비하는 반 자동화 된 접근

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

요약

더 많은 비디오 탐색

이 비디오의 챕터

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

임상 신청

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

HIV-1 감염 CD4의 타고난 감지 평가

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

사람 말초 혈액의 Immunophenotypic 분석에 대한 항체 칵테일을 준비하는 반 자동화 된 접근

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

madSTORM을 이용한 T 세포의 고해상도 이미징