이 프로젝트는 NIH NIAID 계약 번호 HHSN2662000500021C에 의해 지원되었다. 우리는 그의 기술 지원 밍 첸 감사합니다.

1. 프로그램 Amaxa 96 잘 셔틀 Nucleofector <ol><li> 새 매개 변수 파일을 엽니다. </li><li> 당신이 96 잘 플레이트 그림 위에 커서를 드래그하여 표준 transfection에 사용됩니다 웰스의 번호를 선택합니다. 각 실험 샘플에 대해 수영장 3 웰스의 최소 사용합니다. </li><li> 입력 프로그램 코드 : part1 선택 &#39;FF&#39;와 파트에서 선택한 &#39;168 &#39;메뉴를 풀다운에서 </li><li> 솔루션 박스 선택 &#39;단핵구, 인간&#39;에서 </li><li> 제어 옵션에서 &#39;표준&#39;을 선택합니다. </li><li> 적용을 클릭합니다. </li><li> 노 transfection 컨트롤을 포함하려면 필요하고 제어 옵션에서 &#39;없음 프로그램 제어&#39;를 선택하지하고 적용을 클릭하면 다이어그램에서 더 이상 우물을 선택합니다. </li><li> 플레이트 다이어그램에 남아있는 사용하지 않는 우물을 선택하고 Undefine을 클릭합니다. </li></ol> 2. transfection에 대한 DC가 준비 <ol><li> 모든 작업은 가능한 세포 배양 후드에서 무균 조건 하에서 수행되어야합니다. 추가하여 nucleofection 솔루션을 준비 96 - 잘 450-2025의 비율에 인간 단핵구 96 잘 Nucleofector 솔루션을 보완. 믹스 룸 온도 따뜻한 수 있습니다. 당신은 당 잘 nucleofection 솔루션의 20μl가 필요합니다. 오류를 pipetting 수 있도록 10 %의 초과하십시오. </li><li> 당신은 행 1과 2에 첫 번째를 삽입 올바른 방향 IE에서는 nucleocuvette 판에 필요한 nucleocuvette 모듈의 개수를 놓습니다. </li><li> 실험을 위해 10 분 400g에 centrifuging에 의해 잘하고 펠렛 당 500,000 세포에 대한 계산에 필요한 DC가의 수를 결정합니다. 조심스럽게 뜨는을 제거합니다. </li><li> 부드럽게 몇 번 아래 pipetting과로 nucleofection 솔루션에있는 세포를 Resuspend. </li><li> 특정 치료 예 글로 하는걸 - I에 대한 분류 eppendorfs로 resuspended 세포의 올바른 볼륨을 나누십시오. </li><li> pipetting하여 세포와 혼합 500,000 당 0.25μg siRNA를 추가합니다. 당신의 노 transfection 컨트롤 샘플이 아닌 타겟 siRNA를 사용합니다. </li><li> 귀하의 실험 레이아웃에 따라 nucleocuvette 모듈로 위의 혼합물의 피펫 20μl는, 액체를 보장하는 것은 우물의 바닥에 배달됩니다. </li><li> 뚜껑과 nucleocuvette 판을 차지하고 있으며 공기 거품의 제거를 보장하기 어려운 표면 몇번에 번호판을 누릅니다. </li></ol> 3. Transfect DC가 <ol><li> Nucleofector 96 - 웰 셔틀 트레이에 접시를 넣고 업로드를 클릭한 다음 시작합니다. </li><li> 무릎 톱 디스플레이에 transfection 과정의 진행률을 따라, 빨간색 배경에 검은 막대가 실패했습니다 의미 반면 녹색 배경에 검은 십자가, 그 우물에 성공적으로 transfection을 의미합니다. </li><li> transfection 과정의 완료에 접시를 제거하고 잘 멀티채널 피펫을 사용하여 각 DC 성장 매체의 80μl를 추가합니다. </li><li> 37 10 분 번호판을 품어 ° C와 5% CO 2. </li><li> 올바른 방향을 유지, 미리 예열 DC 성장 매체의 100μl를 포함하는 행렬 튜브로 nucleocuvettes에서 100μl 볼륨을 전송합니다. </li><li> transfection이 발생하지 않았 이러한 튜브를 제거하고 폐기. </li><li> 37 품어 ° C와 5% 24 시간 또는 기타 원하는 시간 간격에 대한 CO 2. </li></ol> 4. NDV와 세포를 감염 <ol><li> eppendorfs에 각 실험 샘플에 대한 인큐베이터에서 세포 배양 후드와 수영장 튜브에 매트릭스 튜브를 제거 </li><li> 펠렛 세포가 부드럽게 5 분 데스크 톱 원심 분리기의 회전에 의해하고하면 뜨는 제거합니다. </li><li> 뫄 1에 NDV를 포함하는 혈청없는 성장 매체에있는 세포를 Resuspend 37에서 품어 ° C와 느슨하게 살균 방식으로 덮여 eppendorfs와 5% 45 분에 대한 CO 2. </li><li> 80-10 H.위한 DC 성장 매체 900μl, 다시 품어 추가 </li></ol> 5. 수확 세포 <ol><li> 데스크 톱 원심 분리기에 eppendorfs 잔을 돌리면서 뜨는 제거하여 세포 펠렛. </li><li> 귀하의 프로토콜에 따라 RNA 또는 단백질 추출을위한 수확 세포. </li></ol> 6. 대표 결과 : 우리 최적화된 프로토콜을 사용하는 우리와 DC가를 transfected RIGI 타겟팅 인터페론 반응 경로를 자극하는 NDV (릭 - I에 의해 감지 paramyxovirus)으로 세포를 감염 후 24 H에 대한 siRNA를합니다. qRT - PCR 분석에 의해 우리는 전사 수준에서 75 % 유전자의 다운 노크 보여주었다. 우리는 또한 IFN 신호 계단식 찾으 - I의 스트림 이펙터입니다 IFNβ의 표현에서 유사한 감소를 관찰했다. 또한, 우리는 MxA, IFNβ 하류 응답 유전자의 그 (그림 1A)을 최소화하는 동안 비 감염 제어 transfected 세포에서 IFNβ의 표현이 감지되지 않았음을 관찰했다. 와 DC가의 두 번째 transfection RIGI 타겟 siRNA 것은 서양 얼룩 분석을 포함하는 충분한 세포를 사용하여 수행되었습니다. 어디RT - PCR 결과와 유사했다 우리가 (62 % 및 66% 장비 - I와 IFNβ 표현은 각각의 허물고) 이전에 본 것은 (그림 1B), 서양 얼룩이 유전자의 표현이되어 있다고 발표 전 - 릭에 대한 탐지 완전히 (그림 1C) 차단. <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="그림 1A"/> 그림 1. (A) MoDCs이 중 siRNA 타겟팅 transfected되었습니다 장비를 - I 또는 qRT - PCR에 의해 결정 릭 - I, IFNβ과 MxA의 표현에 특이 현상이 글로의 siRNA 및 효과. (-) transfection 프로토콜은 단핵구 특정 버퍼와 nucleoporation 프로그램 FF168 (Lonza 워커스빌 주) 24 H에 대해 다음과 부화를 사용, transfected 세포는 어느 NDV (+) 또는 왼쪽으로 감염되지 않은 감염되었습니다. 10 H에 대한 자세한 부화 후, 세포는 수확 및 RNA 추출 성적 수준은 두 복제 실험의 결과를 대표했다. 보울스 외 16 일부터 촬영. (B) 그림 1A에 사용되는 같은 다른 버피의 코트에서 얻은 MoDCs 다시와 transfected되었습니다 중 릭 - I - 타겟 siRNA 또는 위와 같은 transfection 프로토콜을 사용하여 특이 현상이 글로의 siRNA합니다. untransfected MoDCs를 사용하여 추가적인 제어 실험에 통합되었습니다. 모든 세포가 NDV 감염과 RNA 및 단백질 추출 모두 수확되기 전에 추가로 10 H에 대한 incubated 있었다 24 H 보육에 따라. 장비 - I, IFNβ 및 qRT - PCR에 의해 결정으로 MxA의 성적 수준은 두 복제 실험의 결과를 나타냅니다. 보울스 외 16 일부터 촬영. (C) 위의 그림 1B에 설명되어있는 세포에서 Lysates는 서양 얼룩에 의해 분석되었다. 레인스 1과 2는 untransfected 세포에서 lysates, 차선 3과 4는 글로의 siRNA transfected 세포에서 lysates, 차선 5 6 장비 - I - 타겟 siRNA와 transfected 세포에서 lysates. 샘플은 장비 - I에 대한 탐지 또한 GAPDH (로딩 제어 등)과 중복에서 실행 하였다. 보울스 외 16 일부터 촬영.

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관심 없음 충돌 선언하지 않습니다.

나이브 기본 돌기 세포의 transfection 효율이 높은 처리량 분석 및 본래 - 적응 면역 전환을 중재이 핵심 세포 세포 염증 경로의 리버스 엔지니어링을 위해 중요합니다. 그러나, 대부분의 조사는 이러한 세포가 표준 transfection 기술을 사용하는 경우 모두 효율적으로 transfection 절차 유도 세포 성숙하지 않고 transfect하기 어려운다는 것을 찾을 수 있습니다. 우리는 이러한 제한이 동시에 독립 96 - 웰 상?...

<table border="1"><tbody><tr><th> 장비 / 시약 </th><th> 회사 </th><th> 카탈로그 # </th><th> 댓글 </th></tr><tr><td> Amaxa Nucleofector 96 - 웰 셔틀 </td><td> Lonza </td><td> 108S0109 </td><td> 일련 번호 </td></tr><tr><td> Amaxa 인간 단핵구 96 - 웰 Nucleofector 키트 </td><td> Lonza </td><td> VHPA - 2007 </td><td> 인간 단핵구 96 - 웰 Nucleofector 솔루션, 96 잘 보충하고 Nucleocuvettes과 접시를 포함 </td></tr><tr><td> 장비 - I siRNA </td><td> Dharmacon </td><td> L - 012511-00 </td><td></td></tr><tr><td> 글로의 siRNA </td><td> Dharmacon </td><td> D - 001600 - 01-20 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11,875 </td><td> 10% FCS와 보충이 MM L - 글루타민, 100 U / ML 페니실린 및 DC 성장 매체를 100 μg / ML 스트렙토 마이신 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11,965 </td><td></td></tr><tr><td> L - 글루타민 </td><td> Invitrogen </td><td> 25030081 </td><td></td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Invitrogen </td><td> 15070063 </td><td></td></tr><tr><td> 소태아 혈청 </td><td> HyClone </td><td> 3070.03 </td><td></td></tr><tr><td> 돌기 세포 </td><td> 뉴욕 블러드 센터 </td><td></td><td> DC가이 표준 절차를 사용하여 버피의 외투에서 정화 아르 </td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

돌기 세포 (DCS)은 자사의 개시 및 감염 1 반응에서 중요한 역할을 면역 시스템의 센티넬 간주될 수 있습니다. 나이브 DC가에 의해 병원성 항원의 검색은 병원체 - 관련 분자 패턴 (PAMPS)로 불리는 특정 보존 구조를 인식할 수있는 패턴 인식 수용체 (PRRs)를 통해이다. DC가로 PAMPs가 탐지 성숙 DC가 자신의 활성화 및 변화의 결과로 세포내 신호 폭포를 트리거합니다. 이 과정은 일반적으로 다른 전염증성 크린 시토킨와 함께 제 1 형 인터페론의 생산이 특징입니다, 이러한 T 세포와 상호 작용이 적응 면역 반응 2을 시작 배수 림프절에 성숙한 DC의 MHCII와 CD86 및 마이 그 레이션과 같은 세포 표면 마커의 upregulation, 3. 따라서 DC가 그 본래 및 적응 면역 시스템을 연결합니다. 각종 병원균에 DC 응답을 기본 분자 네트워크를 해부하다하는 능력은 이러한 신호 경로의 규제 및 유도 유전자를보다 잘 이해하는 데있어 매우 중요합니다. 또한 전염병 및 종양에 대한 DC - 기반 백신의 개발을 용이하게 도움이 될 것입니다. 그러나, 연구의이 라인은 심각하게 기본 DC가 4 transfecting의 어려움으로 장애가되었습니다. 같은 lentiviral 시스템으로 바이러스 전달 방식은, 일반적으로 사용하지만, 이러한 복잡 성과 바이오 유해 위험 (관련 비용) 5,6,7,8 많은 한계를 가지고있다. 또한, 바이러스성 유전자 제품의 배달 DC가 9,10,11,12을 transduced 사람의 immunogenicity을 증가시킵니다. Electroporation은 혼합 결과 13,14,15와 함께 사용하지만, 우리는 높은 처리량 transfection 프로토콜의 사용을보고하고 결론의 유틸리티를 입증하는 최초되었습니다. 이 보고서에서 우리는 제한된 세포 독성 및 DC 성숙 16 부재와 함께, 인간의 기본 DC가의 높은 처리량 transfection을 위해 최적화된 상용 프로토콜을 요약합니다. Transfection 효율 (GFP 플라스미드의)와 세포 생존 능력은 각각 50 % 이상 70 % 였어요. qRT - PCR은 IFNβ 전혀 upregulation을 증명하지 동안 외과 분석, transfected 세포의 성숙 마커 CD86와 MHCII 표현 증가의 부재를 설립하였습니다. 이 electroporation 프로토콜을 사용하여, 우리는 siRNA와 효과적인 타겟 유전자의 허물고와 DC가 성공적으로 transfection에 대한 증거를 제공하는 장비 - I, mRNA와 단백질 수준 모두에서 핵심적인 바이러스성 인식 수용체 16,17.

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

우리는 세포 성숙을 유발하지 않고 플라스미드 DNA 나 siRNA 중과 기본 인간 단핵구 파생 돌기 세포를 transfecting의 효율적인 방법으로서 최적화된 높은 처리량 nucleofection 프로토콜을 제시한다. 우리는 더 타겟 유전자 mRNA와 단백질 수준 모두에서 릭 - 전의 성공적인 siRNA의 입을 대한 증거를 제공합니다.

세포 성숙없이 돌기 세포의 효율적인 Transfection을 위해 최적화된 프로토콜

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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JoVE Journal

Immunology and Infection

20.2K 조회수.  Mount Sinai School of Medicine. 우리는 세포 성숙을 유발하지 않고 플라스미드 DNA 나 siRNA 중과 기본 인간 단핵구 파생 돌기 세포를 transfecting의 효율적인 방법으로서 최적화된 높은 처리량 nucleofection 프로토콜을 제시한다. 우리는 더 타겟 유전자 mRNA와 단백질 수준 모두에서 릭 - 전의 성공적인 siRNA의 입을 대한 증거를 제공합니다.

비디오: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

추적 연구 Qdot Nanocrystals와 Murine 골수 파생 돌기 세포의 생성 및 라벨링

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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면역학 및 감염병학

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

알레르기기도 염증 동안 폐 돌기 세포 성숙과 마이 그 레이션 분석

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

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

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

Nucleofection에 의해 인간의 THP-1 대 식세포의 고효율 형질

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

마우스 수지상 세포 하위 집합의 분리를위한 효율적이고 높은 수율 방법

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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

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

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

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

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

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 ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

생리 적 면역 원 성 수지상 세포 생성을 위한 새로운 프로토콜

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

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 ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

체외 전사 mRNA를 가진 1 차적인 대식세포의 고효율 형질감염

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...