심혈관 질환의 체액 면역 응답 (-이 작품은 독일 Forschungsgemeinschaft (DFG)의 Sonderforschungsbereich Transregio 19 일 (SFB/TR19, C2), 경제 및 기술 프로젝트 ZIM - KF 2727801MD0 연방 교육부 및 혁신 능력의 센터에 의해 지원되었다 ZIK - 하이킹, 교육 및 연구, 독일 연방 교육부의 BMBF FKZ 03Z2CN12). 동물과 주택과 실험은 유럽 연구소 동물 과학 협회 (FELASA)의 연구실 동물 과학 (Gesellschaft 털 Versuchstierkunde, GV-SOLAS)와 제휴를위한 학회의 권고에 따라 수행되었다.

1. 환자 샘플에서 IgG 절연 <ol><li> 빈 Econo 팩 칼럼으로 두 ML 환자의 EDTA (에틸렌 다이아 민 테트라 초산) 플라즈마 당 3 ML 항 IgG 세 파로스를 작성하여 미니 immunoadsorption 항목을 준비합니다. IgG 격리는 환자 플라즈마의 최소한 2 ML이 필요합니다. </li><li> 반 IgG 세 파로스 상단에 필터를 넣고, 열 아래 팁을 열고 잘라 약 1 방울 / 초의 유량에 도달 할 필터를 누르십시오. 열이 부족 보존 솔루션을 보자. </li><li> 3 권 플라즈마의 볼륨 당 NaCl 0.9 %로 열을 씻으십시오. </li><li> 열에서 플라즈마를 피펫. 플라즈마 완전히 반 IgG 세 파로스에 몰두했을 때, 같은 볼륨 0.9 % NaCl로 씻는다. </li><li> 피펫 한 플라즈마 열에서 0.2 M 글리신 HCL, pH를 2.8의 볼륨과 세 파로스로 잠그다 보자. 열에 글리신 HCL의 또 다른 볼륨을 추가합니다. 15 ML 관을 통해 흐름을 수집하여 0.5 M 트리스 - HCL, pH를 8.0로 7.3으로 산도를 조정합니다. </li><li> 열을 생성하려면3 플라즈마 볼륨 글리신 HCL을 씻는다. 2 권 0.9 % NaCl로 최종 세척 후, 열은 다음 플라즈마 샘플에 사용할 수 있습니다. ° C 최대 4 주까지 8 - 또는 열이 보존 솔루션으로 가득과 4에 저장할 수 있습니다. </li><li> cardiomyocytes에서 사용하기 위해, 수집 된 IgG 분획은 실험 버퍼 (EB)에 대해 dialyzed해야합니다. EB의 준비를 들어, 117 MM NaCl, 2.8 MM KCl, 0.6 MM MgCl 2, 1.2 MM KH 2 PO 4, 1.2 MM CaCl 2, 10 밀리미터 HEPES 및 자기 교반기에 20 밀리미터 포도당 1 L 증류수로 추가하고 pH를 조정 7.3 있습니다. </li><li> 100 kDa의 차단 분자량과 셀룰로오스 에스테르 투석 막 관에 IgG의 일부를 입력합니다. 1 L의 EB에 튜브를 넣고, 4에서 자기 교반기로 천천히 저어 ° C. 8 시간 후, 신선한 EB 3 L로 투석 튜브를 전송하고 4시에 또 다른 20 시간을위한 dialyze ° C. </li><li> inactivat 할 수있는 물 욕조에 57시 30 분 ° C에 대한 투석, 열에게 샘플을 따라전자 보완 요소. 총 IgG 농도를 결정 및 330 μg IgG 각을 포함하는 aliquots를 준비합니다. 측정 (단계 4.3)에 대한 cardiomyocytes에 샘플을 추가 할 때, EB 1 ML에 aliquots를 입력합니다. Undiluted aliquots는 더 이상 사용 할 때까지 -20 ° C에 저장할 수 있습니다. </li></ol> 2. 쥐 Cardiomyocytes의 절연 <ol><li> 칼슘 2 + 무료 절연 버퍼 (IB)의 준비를 들어, 110 MM NaCl, 2.6 MM KCl, 1.2 MM MgSO 4, 1.2 MM KH 2 PO 4, 20 밀리미터 HEPES과 자기를 사용하여 1 L 증류수 11 으 포도당을 추가 교반기. 살균을위한 0.2 μm 병 톱 필터를 통해 실내 온도 및 필터에서 7.4로 산도를 조정합니다. </li><li> thermostated Langendorff 재관류 시스템의 상부 저수지에서 IB의 80 ML을 입력합니다. 37 ° C의 버퍼 온도를 유지하고 95% O 5분의 2 % 30 분에 CO 2와 버퍼를 폭기하는 온도 설정을 조정합니다. </li><li> 약 10,000 무게 - collag의 12,000 U를enase 유형 II, 175 MG 지방산 무료 BSA하고 100 MM CaCl 2 재고 솔루션의 22.5 μl를 포함하는 20 ML IB에 용해. 상단 저장소의 절연 버퍼의 pH를 제어하고 필요한 경우 조정합니다. </li><li> 375 μg / kg 체중의 thiopental 200 G - 175의 Wistar 쥐를 마취시키다. thiopental 솔루션에 2,500 IU의 헤파린을 추가합니다. 그대로 대동맥 활과주의 깊게 thoracotomy, 소비세 마음에와 얼음처럼 차가운 0.9 % NaCl로 전송. 혈액의 마음과 주변 조직과 신선한 0.9 % NaCl로 전송를 청소합니다. 대동맥을 노출, 2-3/sec의 하락 속도를 얻고 캐뉼라에 마음을 첨부 Langendorff 시스템의 흐름 감속기를 엽니 다. </li><li> 까지 남은 피를 씻어 한 방울 / 초의 유량과 3 분에 대한 마음을 Perfuse. Langendorff 시스템의 IB를 순환하기 시작한다. 상부 저수지부터 20 ML 버퍼를 제거 등의 80 ML에 도달하는 데 필요한 많은 버퍼로 collagenase 솔루션과 리필 입력합니다. C를 순환<html> 또 다른 27 분에 ollagenase 솔루션입니다. 유량은 흐름 감속기를 사용하여 한 방울 / 초에서 정기적으로 관리 및 유지 관리해야합니다. </li><li> 심방과 대동맥에서 깨끗한 캐뉼라의 마음을, 이탈 작은 조각으로 썰어. collagenase 솔루션은 낮은 저수지로 실행하도록 허용, 40 ML의 최대 볼륨을 줄이고 더 10 다진 심실을 소화 % - 95 % O가 5분의 2 %의 CO 2 연속 폭기에서 15 분. 그 후 50 ML 튜브에 200 μm의 메쉬 크기 거즈를 통해 세포 현탁액을 필터링합니다. </li><li> 200 μM에게 CaCl 2를 포함하는 10 ML의 IB의 객실 온도와 resuspend 세포에서 2 분에 43 XG에서 세포 현탁액을 원심 분리기 : 3 단계에서 세포의 칼슘 2 + 내용을 복원합니다. 다시 원심 분리기, 500 μM CaCl 2 10 ML IB에서 셀을 resuspend. 최종 원심 분리 50,000 세포 / ML의 밀도에 살균 필터 EB의 cardiomyocytes을 (1.8 참조) resuspend 후. </li></ol>Cardiomyocytes의 &quot;&gt; 3 e_title. Fura-2 염색법 <ol><li> 코트 10 μg의 당 잘 laminin과 완전히 건조 될 때까지 기다리과 4 잘 쏠 수 coverglass. </li><li> 각이 잘 커팅 팁과 micropipette를 사용하여에 cardiomyocyte 정지 1 ML을 입력합니다. 세포가 실온에서 1 시간에 준수 할 수 있습니다. </li><li> 10 일 MG / ML의 μl Fura-2 5 ML의 EB의 주식 솔루션입니다.를 희석 어둠 속에서 착색 솔루션세요. </li><li> coverglass 각도에 착색 솔루션의 피펫 0.5 ML의 버퍼와 무소속의 세포를 벗어. 적당한 흔들림에 따라 10 분에 세포를 배양. 그 후, 착색 솔루션을 벗고 일 ML의 EB를 한 번 세포를 씻어 마지막으로 각도에 0.5 ML의 EB를 채 웁니다. 착색 과정에서 직접 빛을 방지하고 항상 어둠 속에서 스테인드 세포를 유지. </li></ol> 4. 셀 단축 및 CA 2 + 과도의 기록 <ol><li> 역 형광의 m에 쏠의 coverglass를 삽입icroscope는 myocyte 칼슘, 수축성 기록 시스템에 연결되어 있습니다. 물론 처음에 유입 및 유출 관과 맞춤 제작 전극을 배치합니다. 1 ML / 분 EB와 (20 V, 1 Hz에서, 5 밀리 초 시간) 전기 자극을 시작으로 Superfuse cardiomyocytes. 세포가 약 2 분의 자극에 적응 할 수 있도록 허용합니다. </li><li> 분명 결 지속적인 수축과 손상로드 모양의 cardiomyocyte를 선택합니다. 시야의 중심에있는 셀을 위치와 카메라 채널을 엽니 다. 셀 프레임 어댑터를 회전하고 현미경 단계를 이동하여 비디오 영역에서 수평으로 셀을 일정한 방향으로 향하게. </li><li> 초기 셀 단축 및 CA 2 + 과도를 얻기 위해 15 수축 - 가장자리가 동영상 영역의 제어 요소 (그림 2)을 검출 조정, 형광 채널과 기록이 10여십시오. </li><li> 형광 얼룩의 표백 방지하기 위해 현미경 광 채널을 닫습니다. 샘플로 EB에서을 통해 흐름을 전환환자 IgG 1 ML과 3 방향 밸브 및 superfuse의 cardiomyocytes으로 우회. 공기가 잘 도달 전에 펌프를 중지합니다. </li><li> 빛 채널을 열고 또 다른 10 기록 - 급성 세포 응답을 얻기 위해 15 수축합니다. 녹화를 일시 중지하고 다시 조명 채널을 닫습니다. 이 후 녹음 및 5 분 반복합니다. </li><li> 우물의 전극을보십시오. EB와 철저하게 튜브를 청소하고 쏠 수의 coverglass의 다음 잘를 계속합니다. </li><li> 각각 세포 단축 및 CA 2 + &#39;Edge-Length/Length &quot;를 사용하여 IonWizard 소프트웨어와 과도하고&quot;fura 2 숫자는 / 비율을 뺀&#39;창을 분석합니다. 초기 셀 단축 및 CA 2 + 급성의 기준 (BL %에서 최대 H)에 언급 된 피크 높이의 과도, 2 분, 5 분 응답에서 변경 률을 계산합니다. </li></ol>

<ol>
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관심 없음 충돌이 선언 없습니다.

제시 방법은 불분명 출신 DCM 환자에서 기능적으로 효과적인 심장 autoantibodies를 감지 할 수있는 적절한 방법을 제공합니다. 다른 방법에 비해, 캠프 레벨 6에 미치는 영향에 의해 β1-adrenoceptor에 대한 기능을 활성화 항체의 검출을 예를 들어, 우리의 방법은 특정 에피토프의 독립적입니다. 물론 문학에 설명 된 다른 에피토프 독립 assays는 IE 패치 클램프 기술 또는 절연 Purkinje ?...

<table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Immunosorba 보존 솔루션 </td><td> Fresenius 의료 </td><td> 903609211 </td><td></td></tr><tr><td> Collagenase 2 형 </td><td> 셀 시스템 </td><td> LS004176 </td><td></td></tr><tr><td> BSA, 지방산 무료 </td><td> 시그마 - 알드리치 </td><td> A6003 </td><td></td></tr><tr><td> Laminin </td><td> 시그마 - 알드리치 </td><td> L2020 </td><td></td></tr><tr><td> Fura-2 AM </td><td> 시그마 - 알드리치 </td><td> F0888 </td><td> DMSO 1 MG / ML </td></tr><tr><td> Econo 팩 크로마토 그래피 열 </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> 스펙트럼 / 포 바이오 셀룰로오스 에스터 투석 Membran전자 </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 네 잘 쏠 수 Coverglass </td><td> Nunc </td><td> 155383 </td><td></td></tr><tr><td> Myocyte 칼슘과 수축성 기록 시스템 </td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6.0 분석 소프트웨어 </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

확장 성 심근증 (DCM)은 젊은 성인 1 심장 마비의 주요 원인 중 하나입니다. 독성 물질에 대한 유전 처분 및 박람회가 환자의 약 셋째에서이 질병에 대한 원인을 알려져 있지만, DCM의 출처는 대부분 불분명 남아 있습니다. 이 환자의 상당수에서 심장 epitopes에 대한 autoantibodies가 감지되었으며 질병 2,3의 발병과 진행에 중요한 역할을 의심하고 있습니다. 심장 autoantibodies의 중요성은 immunoadsorption 3-5로 autoantibodies의 제거 후 DCM 환자에서 관찰 hemodynamic 개선에서 빨간 밑줄이 있습니다. 특정 항원의 다양한 이미 2,3를 확인하고 있으며 이러한 목표에 대한 항체가 immunoassays에 의해 검출 될 수있다. 그러나, 이러한 assays은 자극 (및 따라서 기능적 효과)와 autoantibodies을 차단 구별 할 수 없습니다. eviden 명이 증가이 구별이 중요한 6,7입니다 CE. 또한 cardiotropic 항체의 수에 대한 목표는 여전히 미확인이며, 따라서 immunoassays에 의해 감지 할 수 있다고 가정 할 수 있습니다. 따라서, 우리는 각각의 항원과는 독립적 기능 활성화 cardiotropic 항체의 검출하는 방법을 설립했습니다. 방법의 배경은 일반적으로 포유 동물 8,9 사이에 심장 단백질의 기능 영역에 대한 관찰 높은 호몰 로지입니다. 이 인간의 항원에 대한 감독이 심장 항체 성인 쥐 cardiomyocytes에 DCM 환자에서 IgG의 테스트를 할 수 있습니다 이외의 인간 대상 세포와 함께 상호 반응 제안한다. 우리의 방법은 3 단계로 구성 : 첫 번째는 IgG는 immunoadsorption 열 (PlasmaSelect, Teterow, 독일)에서 얻은 세 파로스 결합 항 IgG 항체를 사용하는 환자의 혈장에서 분리됩니다. 둘째, 성인 cardiomyocytes는 AP를 사용하여 Langendorff 재관류 장치에 collagenase 재관류에 의해 고립되어rotocol은 이전 작품에서 10,11을 수정했습니다. 획득 cardiomyocytes는 laminin 코팅 쏠의 coverglasses에 부착 Fura-2, 쉽게 (CA 2 +) 내용 12 세포 내 칼슘을 관찰하는 셀에 반입 할 수있는 칼슘 선택성 형광 염료를 물들입니다. 마지막 단계에서, 세포 단축 및 CA 2 환자 IgG의 효과 + 필드에 자극을 cardiomyocytes의 과도는 표준 역 형광등에 연결된 상업 myocyte 칼슘, 수축성 모니터링 시스템 (IonOptix, 밀튼, MA, USA)를 사용하여 온라인으로 감시하고 있습니다 현미경.

분야에서 세포 inotropy의 측정을위한 두 예제는 아래에 myocyte 칼슘 2 + 및 수축성 기록 시스템을 사용하여 성인 cardiomyocytes을 (그림 2) 자극. 그림 3은 제어 측정의 인상을줍니다, 그림 4에있는 동안 cardiodepressive 항체의 효과 있는 환자의 DCM가 표시됩니다. 에서 두 예, 초기 세포 단축 및 CA 2 + EB있는 superfusion 동안 과도은 분석...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

제시 방법은 절연 쥐 cardiomyocytes의 휴대폰 단축하고 세포 내 칼슘 과도에 절연 환자의 면역 글로불린의 영향을 분석하여, 관계없이 특정 항원의, 확장 성 심근증 환자의 혈장에 기능을 효과적으로 cardiotropic autoantibodies를 감지 할 수있는 방법을 제공합니다.

절연 Cardiomyocytes의 세포 기능에 대한 항체 효과 측정

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K 조회수.  University Medicine Greifswald. 제시 방법은 절연 쥐 cardiomyocytes의 휴대폰 단축하고 세포 내 칼슘 과도에 절연 환자의 면역 글로불린의 영향을 분석하여, 관계없이 특정 항원의, 확장 성 심근증 환자의 혈장에 기능을 효과적으로 cardiotropic autoantibodies를 감지 할 수있는 방법을 제공합니다.

비디오: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Cytosolic CA의 측정 2 + 절연 수축성 Lymphatics에

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

연구

면역학 및 감염병학

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

유기 용매를 사용하지 않고 트리클로산의 효과 : RBL-2H3 비만 세포 탈과립에 화학 효과를 평가하는 마이크로 분석 실험

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

적혈구에 대한 항체에 의한 보체 활성의 정량 검출하는 방법

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

흐름에서 백혈구의 모집에 기질 세포의 영향 분석

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

펩타이드 모노클로 날 항체 인식 선형 B 세포 에피토프의 확인을 스캔 보조

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

복 포도 당 내성 검사, 폐 기능 측정 및 폐의 고정 비만 및 장애인된 대사 폐 결과에 미치는 영향 연구

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Murine 골 복 막 대 식 세포 활성화에 대 한 항 체 기반 약물 효과의 평가

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

쥐에 있는 침 샘 기능의 측정 하는 방법

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

합성 살 균 소의 건설 맞춤형 다양성 팹 라이브러리 표시

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...