이 작품은 NIH (N01 HHSN272201100019C, U19 AI 089,992, 그리고 NCRR / GCRC 프로그램 M01-RR00125)에 의해 부분적으로 지원되었다. 저자는 더 경쟁 재정적 이익을 선언하지 않고 박사 마크 Shlomchik, 예일 세포 분류기 핵심 시설의 이사 감사합니다.

1. 면역 세포의 분리 및 자극 <ol><li> 지역 기관 검토위원회의 지침하에 작성된 정보에 동의 후 건강한 자원자에서 10-50 ML heparinized 혈액을 구합니다. 노화 또는 질병의 연구를 위해 각 자원이나 환자에 대한 pathogenesis, 배제와 포함 조건은 명시적으로 결정되어야합니다. </li><li> CPT 튜브 또는 Ficoll-Paque을 사용하는 플러스 격리 7 (시약, 표 1) 당일 제조 업체의 지침 (GE 헬스케어, 뉴저지)과 행동 실험에 의하면 인간의 말초 혈액 mononuclear 세포 (PBMCs) 분리. </li><li> 품어 PBMCs (3 × 10 6 / 튜브) 혼자서 0.6 ML 매체에서 1.5 ML 튜브 또는 TLR의 리간드 또는 다른 활성 요원 예로 자극 TLR7 / 8 리간드 R848 (0.6 ML 매체 추가로 10 MM TLR7 / 8 리간드의 0.6 μl 10-60 분 (InvivoGen, CA) 9 37 ° C에서 R848, 최종 농도 10 μm의)가. </li></ol> 2. 연구소세포 eling <ol><li> 라벨 세포 혈통과 fluorescently - 복합 항체를 사용하여 라이브 세포 현탁액의 다른 표면 마커. 라벨 3 * 10 4시 20 분 6 PBMCs ° 단핵구 또는 DC lineages (표 2) 특정 표면 항체의 칵테일과 1.5 ML Eppendorf 튜브에 PBS / 2퍼센트 FBS를 100 μl의 C #. 최적 항체 dilutions이 많은 - 투 - 많은 편차를 최소화하기 위해 일괄적으로 처리되어야 예비 실험 및 샘플을 결정하실 수 있습니다. 0.1의 공급 업체 항체 주식 MG / ML은 항체의 5 μl (1시 20분 희석)을 사용합니다. 악기에 형광 채널을 설정하는 단 하나의 컬러 켤레축으로 표시 셀 별도의 튜브를 사용하십시오. </li><li> 표면 라벨 후에 RT에서 10 분 동안 4% PFA / PBS로 세포를 고정시킨다. 스토리지에 대한 분석 시간까지, 검정 당일까지 -80시 동결 버퍼 500 XG와 resuspend 세포 (90% FBS가 DMSO 10 % 포함) ° C에서 원심 세포. </li><li> 평가, 공정 배치 O에 대한기증자의 그룹에서 f를 치료하고 세포를 자극 함께 다양성​​을 최소화합니다. 분석의 날, 해동 세포를 신속하게 37에 ° C 물 목욕, 동결 버퍼를 제거하는 500x g에서 원심 분리기. </li><li> 세포내 크린 시토킨 또는 기타 마커, 100 μl BD의 파마 / 워시 버퍼 (BD Biosciences, 뉴저지)에서 resuspend 세포 현탁액의 탐지를 위해 세포를 permeabilize합니다. 예를 들어, 20 RT에서 분, 500 XG에서 원심 분리기, 기음 뜨는 및 용 토끼 방지 NF-κB (p65) 항체의 1시 20분 희석 (최종 농도가 10 μg / ML, SantaCruz 생명 공학, CA)과 세포 내 신호 전달 구성 요소의 라벨 100 μl BD의 파마의 resuspend 세포가 / 염소 항 토끼 IgG-Alexa647 (Invitrogen, CA)를 1:250 희석를 포함하는 버퍼를 씻고 RT에서 20 분 동안 품어. </li><li> 영상으로 바로 전에, DAPI (0.2 μg / ML, Invitrogen, CA) 또는 propidium 요오드화물의 (; 20 NG / ML, Invitrogen, CA PI)와 핵 counterstain. </li></ol> 3. ImageStream 분석 <ol><li>인간의 샘플 사이의 다양성을 줄이기 위해 일괄 샘플에서 ImageStream하여 이미징을 실시. 488 nm의, 658 nm의 및 405 nm의 250 MW 10 MW 200 MW 다음과 같이 레이저의 전원을위한 악기 설정했다. </li><li> 파편 (낮은 PI 강도) 또는 다세포 행사에서 단일 세포 (R1) (구별 : (높이 비율 폭) 데이터 첫번째 파일 (그림 1) 정상적인 PI 강도 높은 PI의 화면 비율과 이벤트 게이트를 분석하려면 높은 PI 강도와 낮은 비율). Monocytes은 CD14 양성 세포에 대한 게이트 안에 있습니다. mDCs은 린 1 마커 (R3)의 CD11c과 낮은 CD4 (R2)에 있으며 낮은위한 높다 전지 게이트 안에 있습니다. </li><li> 각 세포의 활성화 정도의 효율적인 양적 척도를 제공하기 위해 (Amnis, WA) 아이디어에게 소프트웨어를 사용합니다. 핵 (R4)에 translocation을 나타내는 세포질 전사 인자 핵 염색 PI와 NF-κB의 유사성 (혹은 공동 지방화)를 결정합니다. NF-κB/PI locali의 높은 상관 관계zation는 높은 유사성 점수에 반영 및 활성화 10 정도를 나타냅니다. </li><li> 다른 치료 그룹 또는 세포의 상대적인 기능적 효율성을 나타내는 환자 집단 사이의 유사성 비율을 비교하십시오. 절대 값 또는 배 차이의 통계적 비교를 통해 샘플간에 데이터를 계량. 인구 histograms의 핵심 세그먼트 (그림 2)에서 세포의 이미지를 표시하는 아이디어 소프트웨어를 사용합니다. </li></ol> 4. 대표 결과 우리는 TLR7 / 8 리간드, R848 (그림 1)과 PBMCs을 자극하여 모델 바이러스성 리간드에 반응 신호 전달 경로를 계량합니다. 우리는 우리의 샘플 그룹 (그림 2)에서 셀룰러 현지화 이미지 및 인구 통계를 모두 수집했습니다. ImageStream 우리 게이트 셀 하위 집합에 직접 PBMCs과 이미지 세포 응답의 문이 있지만, 정렬되지 않은 세포 집단에서 가능합니다. 여기 TLR s의 효과를 계량에서 NF-κB (p65)의 핵 지방화에 ignaling Lin1-, CD4 흐린, CD11c + 골수양 DC가 (mDCs). 베이스 라인에서, 우리가 세포질에서 전사 인자 NF-κB (p65)와 unstimulated 세포의 높은 비율을 관찰했다. 자극 후 치료 전지는 NF-κB (p65)가 (중간 유사성 점수는 각각 1.52과 3.01이며, 그림 자극 후 핵으로 translocated 것을 나타내는 핵 얼룩 PI와 NF-B의 상당히 높은 유사성 점수 (p65)가. 2). 비슷한 결과가 TLR5 - 자극 monocytes 9 설명되어 있습니다. <img alt="그림 1" src="/files/ftp_upload/3741/3741fig1.jpg" /> 1 그림. 염색법과 인간 mDCs에 자극 효과를 계량하기위한 게이팅 전략. NF-κB의 전사 인자는 여러 면역 체계 유전자의 표현을 규정합니다. 이 연구 TLR7 / 8 리가 끝나고 대표 과목에서 mDCs에서 NF-κB (p65)의 핵 지방화를 측정<html> 차 자극. 포커스에 하나의 세포는 높은 핵 가로 세로 비율 (R1)와 PI 긍정적인 사건에 게이팅에 의해 확인되었다. mDCs (Lin1-, CD4dim, CD11c +)는 R3의 문이 있었다. NF-κB (p65)의 핵 지방화는 R4로 꾸몄다됩니다. <a href="http://www.jove.com/files/ftp_upload/3741/3741fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> . <img alt="그림 2" src="/files/ftp_upload/3741/3741fig2.jpg" /> 그림 2. . 자극 후 mDCs에서 NF-κB의 Translocation은 자극 (R4) 후 핵에 NF-κB (p65)의 translocation은 치료와 자극 mDCs의 인구에 묘사된되며 평균 유사성 점수는 치료 예제에서 1.52와 3.01에있다 자극 표본 (). 치료 혹은 R848-자극 세포 집단으로 동시에 수집된 디지털 이미지는 대표 세포와 세포 핵 (B)로 translocated NF-B의 강도를 보여줍니다._upload/3741/3741fig2large.jpg &quot;대상 =&quot;_blank &quot;&gt; 큰 그림을 보려면 여기를 클릭하십시오.

<ol>
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관심의 어떠한 충돌 선언 없습니다.

translational 연구를위한 ImageStream의 사용에서 중요한 단계는 관련 비교 그룹 및 최적화 및 항체 특이성의 검증의 선택입니다. 분류 대상에서 대상 그룹 간의 차이점 histograms 및 세포 영상을 통해 예가 될 것입니다. 관련 수용체 및 신호 전달 경로의 변화에​​ 대한 철저한 분석과 결합된 이러한 데이터는 전문적인 동료에 면역 dysregulation을 기초 메커니즘에 귀중한 통찰력을 제공할 것입니다. 기술?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Ficoll-Paque 플러스 </td><td> GE 헬스케어 </td><td> 17-1440-03 </td></tr><tr><td> R848 </td><td> Invivogen </td><td> tlrl-r848 </td></tr><tr><td> Paraformaldehyde (16 %) </td><td> 전자 현미경 과학 </td><td> 15,710 </td></tr><tr><td> BD는 파마 / 쉐이브 </td><td> BD Biosciences </td><td> 554,723 </td></tr><tr><td> CD14-PE </td><td> eBioscience </td><td> 12-0149-41 </td></tr><tr><td> CD4-PacBlue </td><td> BD Pharmingen </td><td> 68,436 </td></tr><tr><td> 리니지 칵테일 1 (린 1)-FITC </td><td> BD Biosciences </td><td> 340,546 </td></tr><tr><td> CD11c-PE </td><td> BD Biosciences </td><td> 347,637 </td></tr><tr><td> CD123-PE </td><td> BD Biosciences </td><td> 340,545 </td></tr><tr><td> 토끼 반 NF-κB (p65) </td><td> SantaCruz </td><td> SC-372 </td></tr><tr><td> Alexa647 F (AB &#39;) 2 염소 항 토끼 IgG </td><td> Invitrogen </td><td> A21246 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> ImageStream X </td><td> Amnis </td><td></td></tr></tbody></table> 표 1. 특정 시약 및 장비. <table border="1"><tbody><tr><td> 셀 유형 </td><td> FITC </td><td> PE </td><td> 알렉사 647 </td><td> PacBlue </td></tr><tr><td> 단핵구 </td><td></td><td> CD14 </td><td> NF-κB (p65) </td><td></td></tr><tr><td> MDC </td><td> Lin1 </td><td> CD11c </td><td> NF-κB (p65) </td><td> CD4 </td></tr><tr><td> PDC </td><td> Lin1</td><td> CD123 </td><td> NF-κB (p65) </td><td> CD4 </td></tr></tbody></table> 신호 경로의 세포 계보 (Lineage) 염색법에 대한 표 2. 항체 패널 단핵구 : CD14 + MDC : Lin1-, CD4dim, CD11c + PDC : Lin1-, CD4dim, CD123 + Lin1 (혈통) 마커가 인 경우에는 3 번 CD, CD14, CD19, CD20, monocytes, NK 세포, T &amp; B 세포에 대한 CD56 마커를 포함합니다.

quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood

면역 상태에서 개별 유사 감염에 반응을 파악하고 질병의 심각도 및 결과에 기여. 고령화는 바이러스성 및 세균성 감염으로 증가 자화율와 연관된와 체액에 잘 문서화된 감소뿐만 아니라 세포 매개 면역 반응의 1,2와 백신에 반응을 감소합니다. 우리는 최근에 수신자와 같은 수용체 (TLRs), 미생물 감염 및 트리거 항균 호스트 방어 반응 3 감지 타고난 면역 시스템의 주요 구성 요소에 노화의 영향을 평가했습니다. 건강한 인간 기증자의 큰 일대에서, 우리는 노인에서 해당 말초 혈액 monocytes 표현 및 특정 TLRs 4와 돌기 세포에서 유사한 감소 TLR 수준 및 신호 응답 (DC가)의 기능을 감소 보여줬에 필수적입니다 세포를 항원 제시 타고난 및 적응 면역 5 사이의 결합. 우리는 macrophages에 TLR3의 dysregulation을 보였습니다웨스트 나일 바이러스 6,7와 감염에 대한 반응으로 노인 기증자로부터 DC가에 의한 IFN의 D 낮은 생산. immunosenescence 우리의 이해와 치료 개입 파라마운트 응답 특정 세포 유형에 대한 자세한 이해와 신호 전달의 메커니즘 (들)입니다. FACS 또는 immunoblot에 의해 일차 전지 및 측량 세포 마커 영상을 통해 면역 반응의 전통적인 연구 결과 그러나 이러한 연구는 일반적으로 환자에서 제공하는 작은 샘플 볼륨과 여러에 복잡한 실험실 기술을 수행하는 무능력에 의해 기술적으로 제한되며, 크게 우리의 이해를 전진했습니다 인간의 샘플. ImageStream 동시 고해상도 디지털 영상과 양적 유동세포계측법을 결합하기 때문에 환자 자화율의 효율적인 캡처를위한 contemporaneously 여러 세포 인구의 조사를 용이하게합니다. 여기에서 DC가 ImageStream의 사용을 평가하기 위해 입증 TLR7 / 8인산화 및 면역 반응 8에 대한 중요한 여러 유전자의 전사를 시작하는 주요 전사 인자, NF-κB의 핵 translocation의 활성화 - 중재 증가합니다. 우리는 또한 최근 TLR5 신호와 9 노화에 변경된 면역 반응에 기여할 수 오래된 기증자로부터 monocytes에서 NF-κB 경로의 이전에 인식되지 않는 변경을 보여,이 기술을 사용했습니다.

Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

우리는 (ImageStream 기술의 사용을 설명<a href="http://www.amnis.com/" target="_blank"&gt; www.amnis.com</a&gt;), 당신이 잘 정의된 환자의 동료에서 일차 면역 세포의 세포 메커니즘을 계량하기 위해, 동시 고해상도 디지털 영상과 함께 양적 유동세포계측법을 자랑합니다. 우리의 연구 주제 무리에서 소형 시료의 혈통 특정 세포 반응을 계량하기위한 translational 수사 청사진을 제공합니다.

Monocytes과 인간 혈액의 작은 샘플에서 돌기 세포의 리니지 특정 수신자 같은 수용체 - 매개 시그널링의 정량 이미징

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

quantitative-imaging-lineage-specific-toll-like-receptor-mediated

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JoVE Journal

Immunology and Infection

35.0K 조회수.  Yale University School of Medicine. 우리는 (ImageStream 기술의 사용을 설명 www.amnis.com), 당신이 잘 정의된 환자의 동료에서 일차 면역 세포의 세포 메커니즘을 계량하기 위해, 동시 고해상도 디지털 영상과 함께 양적 유동세포계측법을 자랑합니다. 우리의 연구 주제 무리에서 소형 시료의 혈통 특정 세포 반응을 계량하기위한 translational 수사 청사진을 제...

비디오: Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

수신자 같은 수용체 활성화 레굴라팅에 Neu1 Sialidase 활동 감지

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

연구

면역학 및 감염병학

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

쥐 복막 대 식세포의 분리는 수신자 같은 수용체 자극하면 유전자 발현 분석을 수행하기

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

에 대한 응답으로 인간의 기본 호중구와 단핵구에 의해 항진균 활성의 라이브 영상<em&gt;를했다. 투스</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Phenotypical 및 기능 연구에 대 한 인간의 여성 생식 관에서 수지상 세포의 고립

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

개발 및 검증 磁 단일 분자의 배열 디지털 효소 연결 된 Immunosorbent 분석 결과 인간 인터페론-α에 대 한

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...