We thank Hannah Puzas for assistance with system design and configuration, and Kevin Khovananth for technical advice. Funding for this work was provided by The Hearst Foundations and the Providence Portland Medical Foundation.

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YK, ILG, TLM, WLM, DPH 및 KSB : 없음 경쟁 금전적 이해. ANT는 베크만 쿨터의 직원이며,이 비디오 문서의 주식 공개는 벡크 만 쿨터 후원

말초 혈액의 면역 표현형은 면역 개별 응답에 대한 통찰력을 확보하기위한 매우 중요합니다. 문제는 실험 - 투 - 실험 변동성 1,14을 제어하는 분석의 표준화에있다. 변화 중 하나는 주요 소스 샘플의 인간의 조작에있다. 따라서, 시료 처리의 부분 또는 전체 자동화 실험 간 실험 가변성 1,14 극적인 감소를 용이하게 생각할 수있다. 이 프로토콜에서는, 우리는 전체 혈액의 면역 표현형의 샘플 염색에 대한 2 차원 바코드 판독기가 장착 된 자동화 액체 핸들러를 도입하여 분석 변동성을 최소화하기 위해 성공적인 노력을보고한다. 디자인, 우리의 방법은 다양합니다 그들을 정의하는 추가 &quot;자료 칵테일&quot;을 추가하여 다른 면역 하위 집합을 모니터링 할 수 있습니다. 이러한 서브 세트들은 T 헬퍼 세포, T 조절 세포, B 세포, NK 세포, 수지상 세포 및 단핵구 (원고를 포함준비) 18. 우리는 추가 마커 1을 도입하여 컨소시엄의 추천 항체 패널을 채택. 우리가 유도 항원의 검출에 대한 이러한 채널을 예약하고 싶었다로 세포 집단은, 검출기 화려한 보라색 421 (BV421)에 최소한의 방출과 형광 색소에 접합 된 &quot;자료 칵테일&quot;, 피코 에리 트린 (PE)를 사용하여 식별 및 알로 (APC) 하였다. 이 기능은 면역 세포의 활성화 상태를 모니터링 할 수있는 가장 높은 감도를 보장뿐만 아니라,이 세 가지 형광 색소가 가장 많이 유도 마커에 대한 항체가 결합 될 때 새로운 활성화 마커를 유연하게 숙박 할 수 있습니다뿐만 아닙니다. 따라서, 우리의 방법뿐만 아니라 전체 혈액의 면역 표현형을위한 칵테일 제조에 적합뿐만 아니라 세분화 된 조직 (예를 들면, 종양)에서 발견 한 말초 혈액 단핵 세포와 세포를 포함하는 다른 샘플을 염색하는 데 유용합니다. 성공적인 소개우리의 방법의 duction 특별한 실험 실용 준비의 단계에 대한 관심, 그리고 프로그래밍 및 방법의 실행을 필요로한다. 2 차원 바코드 튜브의 제조는 사람이 읽을 수있는 라벨과 지정된 튜브에 항체의 전송과 라벨을 포함한다. 오류의 도입을 방지하기 위해, 우리는 두 사람과이 작업을 수행하는 것이 좋습니다. 스프레드 시트를 변경 할 때마다, 연구자들은이 방법이 제대로 작동하는지 확인해야합니다. 프로그래밍시 적절한 피펫 팅 방법을 선택하는 것은 성공적인 액체 전송을 보장합니다. LLS는 우리가 P50 또는 P200 전도성 팁을 사용하는 항체 튜브의 바닥에서 침전 된 이물질을받지 않고 피펫 팅을 할 수 있습니다. LLS는 P1000 팁 더 민감하고 때로는 거짓 거품의 존재에 의해 트리거로 LLS는 P1000 팁에 대한 좋은 선택지가되지 않을 수 있습니다. 팁의 가장자리 사이의 공간이 충분하지 않으면 실험 실용 정의의 높이는, 예를 들면, 같은 차선의 액체 전사가 발생할 수있는 각 기기 조정되어야튜브의 바닥. 도 12에 도시 된 바와 같이 &quot;기본 칵테일&quot;및 / 또는 &quot;활성화 칵테일&quot;을 잘 볼 텍싱되지 않은 경우, 그것은 최적의 얼룩을 초래할 수있다. 우리는 시약 (19) 분배와 관련 변동성을 줄이기 위해 다른 방법으로 세포 염색을 위해 동결 건조 된 시약을 사용하는 방법에 대해 생각했다. 그러나, 브릴리언트 바이올렛 염료 고분자 접합체 서로 일으키는 비특이적 신호와 상호 작용하는 것으로 알려져있다. 이와 같이, 동결 건조 된 시약에 두 개 이상의 중합체 접합체 (예 BV421 및 BV650 등) 비 특정 시그널링이 발생할 수 첨가 (예 BV421 + 인구 BV650 배경 신호의 증가) 20. 또한, 동결 건조 된 시약은 새로운 염색 등의 유연성이 부족하다. 그들은 일반적으로 더 비싸고 대량 주문이 필요합니다. 이러한 이유로, 2 차원 바코드 튜브 장착 자동화 액체 핸들러를 사용하여 선택 하였다. 그것은 톤 있지만AKE를 시간을 설정하고 이러한 요인은 생산성 향상과 분석의 재현성에 의해 보상됩니다 장기적으로 악기를 구입하기 위해 선행 투자를 수반합니다. 사실, 몇몇 그룹은 이전에 면역 표현형 또는 유사한 애플리케이션 (21, 22)의 워크 플로우에 자동화 액체 핸들러의 성공적인 통합을보고했다. 유동 세포 계측법 분석을위한 자동화 솔루션은 상용 소스 (FACS SPA III, 자동 칵테일 제조 워크 스테이션 및 FlowStainer)에서 사용할 수 있습니다. 이것은 또한 면역 표현형에 대한 자동화 된 칵테일 준비를위한 큰 필요가 있음을 나타냅니다. 이 기술을 마스터 한 후, 우리는 완전 자동화 된 전체 혈액의 면역 표현형의 개발은 더 실험 - 투 - 실험 변동성을 줄일 수, 심지어 23을 설정 다기관 임상 시험에서 전체 혈액의 면역 표현형이 가능 할 수 있다는 구상. 우리는 이미를 Lyse이었다 사용하기 시작했다시간 조수 용해 및 세척 단계를 자동화합니다. 또한 2D 바코드 튜브 항체의 양과 시약 분배 추적 자동 판정 크게 각각 항체의 재고 및 우리의 방법을 통해 품질 관리를 향상시킬 수 있음을 상상할.

인간의 말초 혈액의 면역 표현형은 면역 세포 하위 집합 1 양적, 질적 변화를 결정한다. 이 작업 및 저항 예측 바이오 마커 에이즈 발견 메커니즘에 대한 통찰력을 제공하고, 복합 면역 요법의 개발을 용이하게한다. 따라서, 면역 표현형의 검증 및 표준화 학술 연구자, 임상 실험실과 산업에 상당한 관심의 영역입니다. 유세포 방법에 의해 말초 혈액의 면역 표현형 현재 혈액 학적 악성 종양 3 및 인간 면역 결핍 바이러스 (HIV) 감염 4,5, 면역 요구에 대한 말초 혈액 신뢰성 면역 표현형 그 때문에 검증 및 표준화 할 사항의 임상 관리에 사용되지만 면역 세포 하위 집합 및 활성화 / 억제 수용체 1,6-8보다 광범위한 범위를 필요로한다. Successful 면역 표현형은 장비 설치 및 세포 얼룩 절차 9,10에 관한 실험 - 투 - 실험 변동성을 최소화하기 위해주의 표준화가 필요합니다. 유동 세포 계측법에 대한 장비 설정 표준화가 잘 전 우려 9,11,12를 해결하기 위해 설립 동안, 면역 세포의 서브 세트 및 활성화 상태의 범위를 제한하지 않고 세포의 염색에 관한 변동을 최소화하는 방법을 불분명. 항원의 수가 증가를 검출 할 때, 너무도 오차 변동은 최적의 시약 분주과 교차 오염에 기인 기회를한다. 응고 전혈 phenoptypic 분석 방법 임상 화학 분석에 사용하기 위해 1980 년대 후반에 설립되었다. 이 같은 방법은 샘플 준비를위한 연구 실험실의 요구를 제공하고 있습니다. 중요한 것은, 임상 실험에 사용 된 항체 칵테일은 통상적으로 덜 복잡하다 아바ilable 사전 역가 및 제조 업체에서 미리 혼합. 미리 혼합 된 항체 칵테일의 단 하나의 전송이 필요합니다. 연구 설정에서 10-16 항체의 항체 칵테일 전형적인. 각각의 칵테일 실험실에 의해 안정성을 검증하거나 각 분석하기 전에 신선한 준비해야합니다. 샘플에 대해 여러 항체 칵테일을 준비 50-80 개별 항체의 피펫, 지루하고 오류가 발생하기 쉬운 둘 다 작업을 수반 할 수 있습니다. 칵테일 제조의 자동화는 적은 오류와 같은 수동 칵테일 준비에 비해 여러 가지 장점을 가지고, 피펫의 정확도를 증가하고, 어쩌면 시약 소모 감소했다. 여기서는 제제를 샘플링 관련 변동성을 최소화하기 위해 면역 표현형의 세포 염색 방법으로 2 차원 (2D) 바코드 리더를 구비 한 자동화 된 액체 핸들러의 성공적인 도입을보고한다.

<table><tbody><tr><td>BD LSRFortessa</td><td>BD Biosciences</td><td></td><td>Flow cytometer</td></tr><tr><td>NXp Span-8 Laboratory Automation Workstation&amp;nbsp;</td><td>Beckman Coulter</td><td>A31839</td><td>Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>Insert, Tube, 11 mm, White, for 1.5 ml Microfuge Tubes (case of 25)</td><td>Beckman Coulter</td><td>373696</td><td>Accessary for 24-Position Tube Rack to accommodate microfuge tubes.&amp;nbsp;</td></tr><tr><td>LLS kit</td><td>Beckman Coulter</td><td>719262</td><td>Attach bottom of the deck in Laboratory Automation Workstation to enable liquid level sensing</td></tr><tr><td>24-Position Tube Rack</td><td>Beckman Coulter</td><td>373661</td><td>Tube rack to hold microfuge tubes for antibody cocktails or blood</td></tr><tr><td>BIOHAZARD AUTOCLAVE BAGS 12 x 24 INCHES RED</td><td>LabMart</td><td>M111416</td><td>Disposable autoclave bags for the Laboratory Automation Workstation</td></tr><tr><td>VisionMate High Speed 2D Barcode Reader</td><td>Thermo Scientific</td><td>AB-1850</td><td>2D barcode reader</td></tr><tr><td>8-Channel Handheld Screw Cap Capper/Decapper</td><td>Thermo Scientific</td><td>4105MAT</td><td>For de-capping and capping 2D barcode tubes&amp;nbsp;</td></tr><tr><td>Microcentrifuge tubes</td><td>Fisher Scientific</td><td>02-681-335</td><td>For making cocktail in or supplying blood speciment to the Laboratory Automation Workstation</td></tr><tr><td>CoolRack XT CFT24</td><td>biocision</td><td>BCS-534</td><td>To hold sample tubes.</td></tr><tr><td>1.0 ml Matrix ScrewTop Amber Tubes in Latch Racks</td><td>Thermo Scientific</td><td>3741AMB</td><td>2D barcode tubes to store fluorescent dye-cojugated antibodies</td></tr><tr><td>Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with Barrier, 1,025 &amp;micro;l</td><td>Beckman Coulter</td><td>987925</td><td>Tips for Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>Biomek Span-8 P50 Tips, Conductive, Pre-sterile with Barrier, 50 &amp;micro;l (Case of 10 racks)</td><td>Beckman Coulter</td><td>B01091</td><td>Tips for Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>Biomek Span-8 P250 Tips, Conductive, Pre-sterile with Barrier (Case of 10 racks)</td><td>Beckman Coulter</td><td>394627</td><td>Tips for Laboratory Automation Workstation&amp;nbsp;</td></tr><tr><td>BD FACS Lysing Solution 10x Concentrate</td><td>BD Biosciences</td><td>349202</td><td>RBC lysis</td></tr><tr><td>Falcon Round-Bottom Polystyrene Tubes</td><td>Fisher Scientific</td><td>14-959-5</td><td>Sample tubes</td></tr><tr><td>Anti-CD45 FITC</td><td>BD Biosciences</td><td>347463</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD3 Alexa Fluor 700</td><td>eBioscience</td><td>56-0038-42</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD4 PerCPCy5.5</td><td>BD Biosciences</td><td>341654</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-TCRgd BV650</td><td>BD Biosciences</td><td>564156</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-TCRab BV786</td><td>BD Biosciences</td><td>563825</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD8 APC-H7</td><td>BD Biosciences</td><td>560179</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CCR7 PE-CF594</td><td>BD Biosciences</td><td>562381</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-CD45RA PE-Cy7</td><td>BD Biosciences</td><td>337167</td><td>Effector T cell panel (base)</td></tr><tr><td>Anti-HLA-DR BV421</td><td>BD Biosciences</td><td>562804</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD278(ICOS) PE</td><td>BD Biosciences</td><td>557802</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD38 APC</td><td>BD Biosciences</td><td>340439</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD279(PD1) BV421</td><td>BD Biosciences</td><td>562516</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD357(GITR) PE</td><td>eBioscience</td><td>12-5875-42</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD137(41BB) APC</td><td>BD Biosciences</td><td>550890</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD69 BV421</td><td>BD Biosciences</td><td>562884</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD314(NKG2D) PE</td><td>BD Biosciences</td><td>557940</td><td>Effector T cell panel (activation)</td></tr><tr><td>Anti-CD134(OX40) APC</td><td>BioLegend</td><td>350008</td><td>Effector T cell panel (activation)</td></tr><tr><td>BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper</td><td>Fisher Scientific</td><td>02-689-6</td><td>For collecting blood</td></tr><tr><td>Albumin, Bovine Serum, Fraction V, RIA and ELISA Grade</td><td>EMD Millipore</td><td>126593</td><td>For flow wash buffer</td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S8032</td><td>For flow wash buffer</td></tr><tr><td>Heparin, sodium salt</td><td>Affimetrix</td><td>16920</td><td>For flow wash buffer</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS), 1x, with Calcium, Magnesium, without Phenol Red</td><td>GE Healthcare Life Sciences</td><td>SH30588.02</td><td>For flow wash buffer</td></tr></tbody></table>

a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood

흐름에 의한 말초 혈액의 면역 표현형은 세포 계측법 질병과 치료 기간 동안 주변 백혈구의 주파수 및 활성화 상태의 변화를 결정한다. 그것은 치료 효과를 예측하고 새로운 치료 표적을 식별 할 수있는 가능성이있다. 전체 혈액 얼룩은 시료 준비 중에 발생할 수있는 유물을 최소화 조작되지 혈액을 사용합니다. 그러나, 전혈 염색은 샘플의 무결성을 보장하기 위해 신선하게 수집 한 혈액에서 수행되어야한다. 또한,이 탠덤 염료의 잠재적 불안정성을 방지하고 선명한 보라색 염료 시약 사이의 상호 작용을 방지하기 위해 같은 날 항체 칵테일을 제조하는 것이 가장 좋다. 따라서, 전체 혈액 얼룩은 내 간 실험적인 변화를 통제하기 위해주의 표준화가 필요합니다. 여기서는 antibod 제조 표준 과정으로 2 차원 (2D) 바코드 리더를 구비 한 자동화 된 액체 핸들러 배포 신고유동 세포 계측법에 대한 Y 칵테일. 항체는 데이터베이스에서 컴파일 된 96 웰 형식 및 내용에 배치 2D 바코드 튜브에 옮겼다. 액체 핸들러는 데이터베이스 내 항체 이름을 참조하여 소스 항체 튜브를 찾을 수있다. 우리의 방법은, 소스 항체 튜브의 위치 조정을 위해 지루하고 제거. 또한 사용자가 용이하게 분석 용 소프트웨어 방법에서, 스크립트 재 기입하지 않고 데이터베이스 수정 등 용도 항체, 부피, 및 대상으로 항체 분배 프로세스 상세의 번호를 변경할 수 있도록 융통성을 제공 하였다. 개념 실험의 증거 분석 세포 계측법 11 색, 17 항체 흐름의 복제 준비에 의해 입증 뛰어난 인터 및 인트라 분석 정밀도를 달성했다. 이 방법론은 분석 유동 세포 계측법에 대한 전체 처리량이 증가하고 상세한 pheno에 필요한 복잡한 항체 칵테일을 매일 준비를 촉진갓 수집 된 항 응고 말초 혈액의 typic 특성.

Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

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

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JoVE Journal

Immunology and Infection

10.7K 조회수.  Providence Cancer Center, Providence Portland Medical Center. 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.

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

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

분자 특성 조합 단일 셀 접근 및 인간의 줄기 및 조상 인구 Immunophenotypic이

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

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유전학

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 ...

면역학 및 감염병학

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

분리는 차별화 및 정량화 인간의 혈액에서 B 세포를 항체가 분비 : ELISPOT을 체액 면역의 기능 판독으로

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

표현형 및 기능 분석에 대한 검색 및 희귀의 농축 항원 특이 적 B 세포

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Immunophenotype 및 Cytokine 생산 대량 Cytometry 통해 주변 전 혈의 단일 세포 분석

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

농축 된 인간 기억 B 세포 집단에서 원하는 특이성을 가진 항체의 선택 및 회복을위한 단세포 스크리닝 방법

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

인간 1차 출처에서 인간 MK의 면역페노티핑 및 세포 분류 또는 헤마토포이틱 전조자로부터 시험관내 분화

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

생물학

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody&#39;s isotype, which correlates with the B cells&#39; secretory activity.

ELISpot Assay를 사용한 항체 분비 B 세포의 기능 평가

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

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Immunology

Immune Systems and Components

Assays and Functional Profiling

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.