우리가 그들의 귀중한 공헌에 대한 K. Nakagaki 박사 H. 이시이 박사 K. 스즈키, A. 야마가타, K. Oofusa 매우 감사하고 있습니다.

1. sGMRα의 생산 및 정화 <ol><li> 중합 효소의 연쇄 반응 (PCR)에 의해 인간의 태반 cDNA 라이브러리에서 sGMRα의 cDNA 확대. </li><li> 그의 태그 다른 PCR에 의해 PCR의 amplicon에 3 &#39;끝에 50 기본 baculovirus의 polyhedrin 엔드의 5&#39; - UTR 순서 27 기본 코딩 RGS를 추가합니다. </li><li> 플라스미드 pMSG1.1MG로 증폭 PCR 제품을 넣습니다. </li><li> 도우미 벡터 누에 쿨러 - W1 스트레인의 계란에 pHA3PIG로 구성 psGMR/M1.1MG을 주사. </li><li> 25 나방에 후면 부화 유충 G0 ° C. 형질 누에가 sGMRα 유전자를 베어링 얻기 위해 눈에 MGFP 표현을 위해 형제간에 또는 쿨러 - W1과 짝짓기로 확인한 화면 G1의 배아. </li><li> 500mM NaCl을 포함하는 인산염 버퍼와 유전자 변형 누에의 고치에서 재조합 sGMRα을 추출하고 4 저어 ° C를 24 시간을위한. </li><li> 이 precipitates 제거하는 15에 대한 20,000그램에서 샘플을 원심 분리기. </li><li> 니켈 - 친화 컬럼에 뜨는 적용됩니다. </li><li> 10mM 이미다졸, 500mM NaCl 및 20mM 인산 나트륨, pH6.3, 그리고 10mM에서 250mM로 졸의 선형 그라디언트로 elute로 컬럼을 씻으십시오. 에 대한 정화 sGMRα 및 dialyze 들어있는 분수 수영장 생리 인산염은 버퍼 (pH7.4) (PBS). </li></ol> 2. GM - CSF 재조합의 Biotinylation <ol><li> PBS에 대한 투석에 의해 sorbitol 준비를 제거합니다. </li><li> 얼음 솔루션 4 20mM 추위 나트륨 메타 periodate 솔루션 ° C의 1ml을 추가합니다. </li><li> 어두운 얼음 속에서 30 분에 대한 샘플을 품어. </li><li> 15mM의 최종 농도의 용액에 글리세롤을 추가합니다. </li><li> 100mM 아세트산 나트륨 버퍼 (pH5.5)에 대한 솔루션을 Dialyze </li><li> 5mM의 최종 농도에서 솔루션에 비오틴의 히드라 지드를 추가하고 지속적으로 실온에서 2 시간에 대한 솔루션을 선동하다. </li><li> PBS, pH7.4에 대한 투석에 의해 비 반응 물질을 제거합니다. </li></ol> 3. 셀 - 무료 GM - CSF 수용체 - 리간드 결합 분석 <ol><li> 4 박 단클론 항 polyHistidine 항체의 50μl와 코트 microtiter 접시 ° C. </li><li> PBST의 500μl와 다섯 번 씻으십시오. </li><li> 4 3H에 대한 차단 솔루션과 부화 ° C.의 100μl 추가 </li><li> PBST의 500μl와 다섯 번 씻으십시오. </li><li> 차단 솔루션에 sGMRα의 50μl를 추가하고 4 박 품어 ° C. </li><li> PBST의 500μl와 다섯 번 씻으십시오. </li><li> GM - CSF 다음 autoantibody와 GM - CSF biotinylated의 25μl를 포함하는 샘플 25μl를 추가합니다. 4 1 시간에 대한 품어 ° C는 sGMRα - GM - CSF의 복합을 형성합니다. </li><li> PBST의 500μl와 다섯 번 씻으십시오. </li><li> 4 1시간에 대한 0.4mM streptavidin 알칼리성 인산 가수 분해 효소의 50μl를 추가 ° C의 biotinylated GM - CSF 바운드 sGMRα를 검색할 수 있습니다. </li><li> PBST의 500μl와 다섯 번 씻으십시오. </li><li> 솔루션에 chemiluminescent 기판의 50μl를 추가하고 실온에서 1 시간을위한 incubated. </li><li> chemiluminescence 플레이트 판독기를 사용하여 chemiluminescence 활동을 감지합니다. </li></ol> 4. 대표 결과 : GM - CSF 수용체 신호 전달 경로의 첫 번째 단계는 세포 표면에 GM - CSF 수용체 알파에 GM - CSF의 바인딩입니다. GM - CSF autoantibody 특별히 GM - CSF를 바인딩하고 체외 16,17에있는 수용체에 그것의 바인딩을 차단하기 때문에, 우리는 autoantibodies 직접 GM - CSF에 바인딩하여 첫 번째 반응을 억제 것을 가상. 마찬가지로 앞에서 설명한 (Ref. 9-15), 우리는 고순도로 재조합 sGMRα 많은 양의 얻기 위해 유전자 변형 누에 기술을 적용. 오직 monomeric 양식이 감소 조건에서 검색되었습니다 반면, 누에 파생 재조합 sGMRα가 아닌 절감 조건 SDS - PAGE에로드되었을 때, sGMRα은 (그림 1B), 두 monomeric (45 KDA)와 dimeric (90 KDA) 양식을 보여주 , 재조합 sGMRα은 단량체 및 체인이 황을 통해 연결된 dimmers 18 혼합 것을 나타냅니다. 셀 - 무료 시스템 (그림 2A)를 사용하여, 우리는 GM - CSF의 autoantibodies (그림 2B 및 그림. 2C)에 의해 sGMRα에 GM - CSF 바인딩의 억제를 평가했다. 이러한 방법은 우라노 외 의해 참조 19 설명했다. 성장 억제는 밀접하게 GM - CSF autoantibody (그림 3A) 19의 다양한 농도에 구속력을 억제 (R = 0.988, P = 0.002)과 상관했다. 마찬가지로, 바인딩 억제가 크게 성장 억제와 상관했다. 바인딩 억제는 GM - CSF의 autoantibodies (그림 3B) 19 복용량에 의존 방식으로 증가했다. 다른 환자에서 혈청 IgG의 분수에 대한 이러한 두 매개 변수가 서로 상관 있었다 (R = 0.589 P = 0.006, 그림. 3C). 어느 구속력을 억제하거나 성장 억제는 KD 가치와 상관하고, 따라서 두 매개 변수를 바인딩 친화력 19 영향을받지되었습니다. 구속력와 g 사이의 데이터의 재현성 결과세 가지 다른 샘플에 세 개의 독립적인 실험을 통해 얻은 rowth 억제가 평가되었다. 변화의 계수는 세포없는 시스템 bioassay (표 1)의 그것보다 더 좋은 것을 지적했다. <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="그림 1" /> 그림 1. 유전자 변형 누에를 사용 sGMRα의 생산을위한 절차 플로우 차트. 변환 벡터 9-11의 A) 구조물. 절감 및 비 절감 조건 정화 sGMRα의 B) SDS - PAGE와 쿠매시 브릴리언트 블루 염색법. sGMRα, 가용성 GM - CSF 수용체 알파, MGFP, 괴물 녹색 형광 단백질, BmNPVpol5&#39; - UTR, Bombyx 모리 핵 polyhedrosis 바이러스 polyhedrin의 5&#39; - 번역되지 않은 지역의 순서, SV40 polyA, SV40 polyA 신호 시퀀스, P 3xP3, 3xP3 발기인; P ser1, ser1 발기인; fibL polyA, 피브로인 L - 체인 polyA 신호 시퀀스, hr3, Bombyx 모리 핵 polyhedrosis 바이러스 hr3 향상제. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="그림 2" /> 그림 2. 전지 - 무료 분석 시스템의 제도. 누에 제작한 sGMRα를 사용하여 A) 경쟁 바인딩 분석. B) 중화가 아닌 중화 항체의 효과 세포가없는 시스템에서 바인딩을 억제에. C) 중화 항체가 아닌 중화 항체의 다양한 농도 사이의 바인딩 억제의 차이. GM - CSF, 과립 세포 대식 세포 콜로니 - 자극 인자, sGMRα, GM - CSF 가용성 수용체 알파, 그의, RGS, 그 태그, AP, 알칼리성 인산 가수 분해 효소. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="그림 3" /> 그림 3. GM - CSF polyclonal 항체 또는 면역 PAP 환자에서 혈청 IgG의 분수의 효과에 의해 sGMRα에 GM - CSF 바인딩을 억제. GM - CSF autoantibody에 의해 구속력을 저해하고 성장 억제 사이의) 관계. GM - CSF autoantibody의 다양한 농도에서 B) 바인딩 억제. 혈청 IgG의 분수 19 %의 바인딩을 억제하고 %의 성장 억제를위한 IC ~ 50 세 C) 관계. IC 50, 50 % 억제 농도, GM - CSF, 과립 세포 대식 세포 콜로니 - 자극 인자. <table border="1"><tbody><tr><td> 견본 </td><td> </td><td> B </td><td> C </td></tr><tr><td colspan="4"> 인터 - 검정 </td></tr><tr><td> # 결정의 </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> 평균 값 (% 구속력을 억제) </td><td> 61.6 </td><td> 63.8 </td><td> 69.7 </td></tr><tr><td> 평균 값 (%의 성장 억제) </td><td> 21.0 </td><td> 73.4 </td><td> 82.8 </td></tr><tr><td> 변동 계수 (%) (% 구속력을 억제) </td><td> 6.0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td> 변동 계수 (%) (%의 성장 억제) </td><td> 64.4 </td><td> 18.3 </td><td> 10.4 </td></tr></tbody></table> 두 assays 5 NG / ML GM - CSF 및 GM - CSF autoantibody와 같은 농도에서 수행되었다. 표 1. 세 독립적인 실험을 통해 얻은 %의 바인딩과 성장의 통제 사이의 변형 계수의 비교.

<ol>
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셀 - 무료 분석 뛰어난 재현성과 인텔리와 GM - CSF의 autoantibodies의 중화 능력을 추정했다. GM - CSF의 autoantibodies 또는 환자의 혈청 IgG의 분수에 의해 바인딩을 억제이 분석에 의해 평가되었다. 데이터 셀없는 분석의 바인딩 억제 각각 TF - 1 세포를 사용하여 bioassay의 성장을 억제 사이의 상관을 보여주었다. bioassay 널리 이용하지만, 여러 가지 시설과 우리는이 새로운 시스템을 사용하여 피할 수 있습니...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

배경 : 이전, 우리가 중화 능력을 보여주지만, GM - CSF autoantibody 아니다 농도가 면역 폐의 폐포의 proteinosis (PAP) 1-3 환자의 질병 심각와 상관했다. 폐에 GM - CSF의 bioactivity 폐기가 4,5 면역 PAP에 대한 가능성이 원인이기 때문에, 그것은 PAP 각 환자의 질병 심각도를 평가하기위한 GM - CSF의 autoantibodies의 중화 능력을 측정하는 약속입니다. 지금까지 GM - CSF의 autoantibodies의 중화 능력은 인간의 골수 세포 또는 GM - CSF 6-8로 자극 TF - 1 세포의 성장 억제를 평가하여 평가되었습니다. bioassay 시스템에서, 그러나, 그것은 지속적인 상태로 세포를 유지 기술적인 문제로 인해 서로 다른 실험실에서 데이터를 비교하기 위해 신뢰할 수있는 데이터뿐만 아니라를 얻기 위해 종종 문제가됩니다. 목표 : 세포없는 수용체 결합 - 검정을 사용하여 세포 표면에 GM - CSF 수용체에 바인딩 GM - CSF를 모방합니다. 방법 : 트렌스 제닉 누에 기술은 고순도 9-13로 재조합 수용성 GM - CSF 수용체 알파 (sGMRα)에 대한 큰 금액을 얻기 위해 적용되었다. 재조합 sGMRα은 실크 단백질에 융합되지 않고 실크 스레드의 친수성 sericin 층에 포함된되었고, 따라서, 우리는 쉽게 중립 수성 솔루션 14,15 잘 순도의 고치에서 추출할 수 있습니다. 다행히도, GM - CSF와 바인딩에 대한 중요한 올리 고당 구조는 다른 곤충이나 효모에 의해 만들어진보다 인간 sGMRα의 구조에 더 비슷합니다. 결과 : sGMRα를 사용하여 셀 - 무료 분석 시스템이 높은 소성과 안정성으로 데이터를 굴복. sGMRα에 바인딩 GM - CSF는 sGMRα를 사용하여 새로운 세포가없는 분석 시스템이 중화 활동의 측정에 대한 더 유용하다는, TF - 1 세포를 사용하여 bioassay와 비슷한 방식으로 polyclonal GM - CSF autoantibody에 의해 저해 약을 - dependently되었습니다 TF - 1 세포 또는 인간의 골수 세포를 사용하여 bioassay 시스템보다 GM - CSF의 autoantibodies니다. 결론 : 우리는 GM - CSF autoantibody의 중화 능력을 quantifying 셀 - 무료 분석을 설립했습니다.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

우리는 수용체에 과립 세포 대식 세포 콜로니 - 자극 인자의 바인딩 (GM - CSF)를 추정하기 위해 셀 무료 수용체 결합 분석을 설계했습니다. 그것은 우수한 재현성과 GM - CSF autoantibody에 의해 용해 GM - CSF 수용체 알파에 바인딩 GM - CSF biotinylated의 경쟁 억제를 평가하는 데 있습니다.

재조합 가용성식이 GM - CSF 수용체를 사용하여 GM - CSF 항체의 중화 능력을 예측 세포 분석 시스템 무료

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

Research

JoVE Journal

Immunology and Infection

12.6K 조회수.  Niigata University Medical and Dental Hospital. 우리는 수용체에 과립 세포 대식 세포 콜로니 - 자극 인자의 바인딩 (GM - CSF)를 추정하기 위해 셀 무료 수용체 결합 분석을 설계했습니다. 그것은 우수한 재현성과 GM - CSF autoantibody에 의해 용해 GM - CSF 수용체 알파에 바인딩 GM - CSF biotinylated의 경쟁 억제를 평가하는 데 있습니다.

비디오: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

인간 백혈구 및 혈소판의 분리 및 평가를위한 프로토콜 : 생물 에너지와 산화 버스트

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

연구

면역학 및 감염병학

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

파지 - 표시 합성 항체 라이브러리의 확장 성 높은 처리량 선택

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

마우스 골수에서 생성 및 GM-CSF의 식별 파생 폐포와 같은 대 식세포와 수지상 세포

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

인플루엔자 바이러스 단일 클론 항 체를 중화의 탈출 변종의 생성

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

사용 하 여 MDCK SIAT1 세포 분석 실험 측정 인플루엔자 항 체 응답 Microneutralization에 의해 인간의 세라에 A(H3N2) 바이러스를 중화

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

생체 외에서 마우스 Granulocyte 대 식 세포 콜로 니 자극 인자 (GM-CSF)-T (THGM) 도우미 셀 생산

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.