이 작품은 장 공 의료 재단 (보조금 번호 CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632, 및 CMRPG3E0633)에 의해 지원 되었다.

연구 기관 검토 위원회의 장 공 기념 병원 Linkuo, 타오 위 엔, 대만 (102-2577A3)에 의해 승인 되었다.
1. NR1 GFP 식 플라스 미드의 준비
<ol><li>살 균 1.5 mL에 대장균 유능한 세포 변형 DH5α의 100 µ L로 NR1 GFP 플라스 미드의 믹스 10 ng까지 부드럽게 튜브를 피펫으로 혼합물을 원심 및 아래쪽 4-6 시간, 20 분 동안 얼음에 튜브를 품 어 고.</li>
	<li>물 욕조에 1 분 동안 42 ° C에 튜브를 품 어 물 탕에서 튜브를 제거, 튜브에 1 mL lysogeny 국물 (파운드) 매체를 추가 하 고 1 시간 동안 흔들어와 인큐베이터에서 37 ° C에서 튜브를 품 어.</li>
	<li>50 µ L 암 피 실린 (0.1 mg/mL)를 포함 하는 파운드 한 천 배지 위에 혼합물의 확산과 하룻밤 인큐베이터에서 37 ° C에서 파운드 천 배지를 품 어.</li>
	<li>멸 균 팁을 사용 하 여 하룻밤 파운드 한 천 배지에서 단일 식민지를 선택 하 고에서 팁을 소용돌이 파운드 매체와 암 피 실린 (0.1 mg/mL) 5 mL를 포함 하는 세균성 문화 관에서. 37 ° c 하룻밤 떨고와 인큐베이터에서 튜브를 품 어.</li>
	<li>Aliquot 3 mL 250 mL 살 균 파운드 매체와 암 피 실린 (0.1 mg/mL)에 포함 된 500 mL 플라스 크에 하룻밤 세균성 문화. 떨고와 37 ° C에서 플라스 크를 품 어. 600의 파장에서 세균성 문화 광학 밀도 (OD)를 정기적으로 확인 nm는 OD600 0.6-1.0에 도달할 때까지 한 분석기를 사용 하 여. 참고: 글리세롤 재고를 준비 하 여 글리세롤 재고 미래 사용을 위한-80 ° C이 하 저장 200 µ L 글리세롤과 잘 800 µ L의 하룻밤 세균성 문화를 혼합.</li>
	<li>NR1 GFP 식 플라스 미드 250 mL 세균성 문화에서 준비<ol>
<li>4 ° c.에 15 분 동안 6000 x g에서 원심 분리 하 여 세포를 수집</li>
		<li>10 mL 물의 resuspension 버퍼 RNase A;를 포함 하는 박테리아 펠 릿 resuspend 와 동 없는 덩어리를 볼 때까지 철저 하 게</li>
		<li>세균 현 탁 액을 10ml 세포의 용 해 버퍼를 추가, 부드럽게 반전 혼합물, 4-6 시간 하 고 lysate 5 분 실 온 (RT)에서 품 어.</li>
		<li>10 mL 미리 냉장된 강수량 버퍼는 lysate를 추가, 부드럽게 반전 혼합 4-6 시간.</li>
		<li>Lysate의 모자와 필터 카트리지를 통에 부 어 실시간에서 10 분 동안 대기</li>
		<li>카트리지 출구 노즐에서 뚜껑을 제거, 카트리지에 플런저를 삽입 하 고 플런저를 조심 스럽게 누릅니다. 수집 하는 필터링 된 50 mL 튜브에 lysate.</li>
		<li>2.5 mL 독점 버퍼 제조 업체에서 하는 필터링 된 lysate를 추가 하 고 약 10 배, 튜브를 거꾸로 하 여 혼합물을 섞어 30 분 동안 얼음에 혼합물을 품 어.</li>
		<li>필터 열을 미리 equilibrated 10 mL 평형 버퍼 필터링된 lysate 혼합물을 적용 합니다. 중력에 의해 배수를 열을 수 있습니다.</li>
		<li>30 mL 워시 버퍼 열 두 번 세척 후 15 mL 차입 버퍼를 사용 하 여 열에서 DNA을 elute.</li>
		<li>10.5 mL (0.7 볼륨) 소 프로 파 놀 eluted DNA 버퍼에 추가, 잘, 그리고 20000 x g 4 ° c.에 30 분 동안에 혼합물을 원심</li>
		<li>신중 하 게는 상쾌한 가만히 따르다, 한 번, 5 mL도 무료 70% 에탄올과 DNA 펠 릿을 세척 하 고 10 분 20000 x g에서 원심.</li>
		<li>상쾌한 가만히 따르다, 화학 후드, 5-10 분 동안 펠 릿 건조 하 고도 무료 버퍼의 300-500 µ L에 펠 릿을 녹.</li>
		<li>분 광 광도 계를 사용 하 여 UV 260/280 nm에 해결책의 흡수를 측정 하 여 플라스 미드 농도 결정 합니다. 참고: 식 플라스 미드 GFP 동일한 프로토콜을 사용 하 여 준비 합니다.</li>
	</ol>
</li>
</ol>2. NR1 GFP 식 플라스 미드와 HEK293 세포의 transfection
<ol><li>각각 2% 젤라틴 솔루션의 aliquot 200 µ L 48-잘 문화 판의 고 적어도 30 분 동안 37 ° C에서 접시를 품 어.</li>
	<li>세포 배양 10% 태아 둔감 한 혈 청 (FBS)에 각 잘 포함 하의 200 µ L에 잘, 그리고 씨앗 5 x 104 HEK293 세포에서 젤라틴 솔루션 발음 37 ° c 하룻밤 5% CO2 와 함께 제공 된 습도 인큐베이터에서 접시를 품 어. 참고: 셀을 hemocytometer를 사용 하 여 계산 했다.</li>
	<li>다음 날, 바꿉니다 소비 문화 중간 10%를 포함 하는 신선한 문화 매체의 200 µ L FBS 잘 당.</li>
	<li>각 단일, 100를 혼합 하 여 솔루션 A를 준비 20 µ L 문화 매체, 및 0.8 µ L transfection 시 약 20 µ L 문화 매체와 혼합 하 여 솔루션 B NR1 tGFP 플라스 미드의 ng. 각 실험에 대 한 전체 볼륨을 준비 하는 데 필요한 우물의 수를 곱하면.</li>
	<li>솔루션 및 솔루션 B, 혼합 하 여 솔루션 C를 준비 하 고 20-30 분에 대 한 실시간에 혼합물을 품 어.</li>
	<li>C 솔루션 필요 각 잘 포함 하는 HEK293 세포의 aliquot 40 µ L 플레이트를 부드럽게, 소용돌이 그리고 하룻밤 5% CO2 와 함께 제공 된 습도 인큐베이터에서 37 ° C에서 접시를 품 어.</li>
	<li>NR1 GFP 40 X-200 X 확대와 함께 형광 현미경 호스트 세포에서 재조합 형 단백질의 표정을 확인 (여기/방출: 482/502 nm), 세포 기반 면역 형광 분석을 진행 하 고. 참고: 동일한 프로토콜을 사용 하 여 HEK293 세포로 식 플라스 미드 GFP Transfect.</li>
</ol>3. 세포 기반 면역 형광 분석 결과
<ol><li>우물에서 보낸된 중간 발음, 다음 세척의 인산 염 200 µ L 각 잘 버퍼링 염 분 (PBS) 3 번.</li>
	<li>각 잘;에 4 %paraformaldehyde 솔루션의 200 µ L를 추가 15 분에 대 한 실시간에 접시를 품 어.</li>
	<li>우물에서 paraformaldehyde 솔루션을 발음 하 고 각각 씻어 PBS의 200 µ L 세 번 잘.</li>
	<li>PBST에 10% 탈지 우유의 200 µ L를 추가 (0.1 %PBS 트윈-20) 솔루션을 각각 잘 고 부드러운 진동으로 1 시간에 대 한 실시간에 접시를 품 어.</li>
	<li>잘에서 PBST 솔루션 10% 탈지 우유를 발음 다음 200 µ L PBST와 웰 스를 한 번 세척.</li>
	<li>1 시간에 대 한 실시간에 부드러운 동요와 1 차 항 체로 희석된 플라즈마 (PBST에 1: 100)와 웰 스를 품 어. 참고: 플라즈마 면역 뇌염이 있기 위하여 의심 되는 환자의 혈액에서 얻은 것입니다.</li>
	<li>우물에서 희석된 플라즈마를 발음 하 고 각각을 씻어 PBST의 200 µ L 세 번 잘.</li>
	<li>염소 안티 인간 IgG 각 음에 알 렉 사 Fluor 594 (1:1, PBST에서 000 희석)와 활용의 200 µ L을 추가 하 고 1 시간 동안 부드러운 흔들어와 RT에서 문화 접시를 품 어.</li>
	<li>IgG 알 렉 사 Fluor 594 솔루션 활용 된 염소 항 인간의 발음 및 각 세척 PBST의 200 µ L 세 번 잘.</li>
	<li>각 잘을 100 µ L 글리세롤 솔루션 (PBS와 1:100,000의 4', 6-diamidino-2-phenylindole (DAPI)에 50% 글리세롤)를 추가 합니다.</li>
	<li>관찰 하 고 형광 현미경 접 안 렌즈, 그리고 4 배, 10 배, 20 X 목표 X 10을 사용 하 여 셀을 이미지. 참고: 각 염료에 대 한 올바른 필터를 사용 하 여: NR1 GFP에 대 한 (여기/방출 = 482/502 nm), 알 렉 사 Fluor 594 대 한 (여기/방출 = 561/594 nm), DAPI에 대 한 (여기/방출 358/461 = nm). 같은 방식으로 표현 하는 GFP HEK 세포를 사용 하 여 부정적인 제어를 수행 합니다.</li>
</ol>

<ol>
	<li>Linnoila, J. J., Rosenfeld, M. R., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+surface+antibody-mediated+autoimmune+encephalitis.">Neuronal surface antibody-mediated autoimmune encephalitis.</a> Semin Neurol. 34 (4), 458-466 (2014).</li><li>Lancaster, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+and+treatment+of+autoimmune+Encephalitis.">The diagnosis and treatment of autoimmune Encephalitis.</a> J Clin Neurol. 12 (1), 1-13 (2016).</li><li>Pruss, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+analysis+of+NMDA+receptor+antibodies+in+encephalitis+of+unknown+origin.">Retrospective analysis of NMDA receptor antibodies in encephalitis of unknown origin.</a> Neurology. 75 (19), 1735-1739 (2010).</li><li>Leypoldt, F., Armangue, T., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+encephalopathies.">Autoimmune encephalopathies.</a> Ann N Y Acad Sci. 1338, 94-114 (2015).</li><li>Dalmau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+encephalitis:+case+series+and+analysis+of+the+effects+of+antibodies.">Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.</a> Lancet Neurol. 7 (12), 1091-1098 (2008).</li><li>Titulaer, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prognostic+factors+for+long-term+outcome+in+patients+with+anti-NMDA+receptor+encephalitis:+an+observational+cohort+study.">Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study.</a> Lancet Neurol. 12 (2), 157-165 (2013).</li><li>Chapman, M. R., Vause, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA+receptor+encephalitis:+diagnosis,+psychiatric+presentation,+and+treatment.">Anti-NMDA receptor encephalitis: diagnosis, psychiatric presentation, and treatment.</a> Am J Psychiatry. 168 (3), 245-251 (2011).</li><li>Gable, M., Glaser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-n-methyl-d-aspartate+receptor+encephalitis+appearing+as+a+new-onset+psychosis:+disease+course+in+children+and+adolescents+within+the+california+encephalitis+project.">Anti-n-methyl-d-aspartate receptor encephalitis appearing as a new-onset psychosis: disease course in children and adolescents within the california encephalitis project.</a> Pediatr Neurol. 72, 25-30 (2017).</li><li>Dalmau, J., Lancaster, E., Martinez-Hernandez, E., Rosenfeld, M. R., Balice-Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+experience+and+laboratory+investigations+in+patients+with+anti-NMDAR+encephalitis.">Clinical experience and laboratory investigations in patients with anti-NMDAR encephalitis.</a> Lancet Neurol. 10 (1), 63-74 (2011).</li><li>Dalmau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraneoplastic+anti-N-methyl-D-aspartate+receptor+encephalitis+associated+with+ovarian+teratoma.">Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma.</a> Ann Neurol. 61 (1), 25-36 (2007).</li><li>Irani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methyl-D-aspartate+antibody+encephalitis:+temporal+progression+of+clinical+and+paraclinical+observations+in+a+predominantly+non-paraneoplastic+disorder+of+both+sexes.">N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes.</a> Brain. 133 (Pt 6), 1655-1667 (2010).</li><li>Suh-Lailam, B. B., Haven, T. R., Copple, S. S., Knapp, D., Jaskowski, T. D., Tebo, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+antibody+encephalitis:+performance+evaluation+and+laboratory+experience+with+the+anti-NMDA-receptor+IgG+assay.">Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay.</a> Clin Chim Acta. 421, 1-6 (2013).</li><li>Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+of+glutamate+receptor+ion+channel+complexes.">Structural biology of glutamate receptor ion channel complexes.</a> Curr Opin Struct Biol. 41, 119-127 (2016).</li><li>Ramberger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+diagnostic+accuracy+of+microscopy+and+flow+cytometry+in+evaluating+n-methyl-d-aspartate+receptor+antibodies+in+serum+using+a+live+cell-based+assay.">Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating n-methyl-d-aspartate receptor antibodies in serum using a live cell-based assay.</a> PLoS One. 10 (3), e0122037 (2015).</li><li>Wandinger, K. P., Saschenbrecker, S., Stoecker, W., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+encephalitis:+a+severe,+multistage,+treatable+disorder+presenting+with+psychosis.">Anti-NMDA-receptor encephalitis: a severe, multistage, treatable disorder presenting with psychosis.</a> J Neuroimmunol. 231 (1-2), 86-91 (2011).</li><li>Dahm, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seroprevalence+of+autoantibodies+against+brain+antigens+in+health+and+disease.">Seroprevalence of autoantibodies against brain antigens in health and disease.</a> Ann Neurol. 76 (1), 82-94 (2014).</li><li>J&#233;z&#233;quel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-+and+single+molecule-based+methods+to+detect+anti-n-methyl-d-aspartate+receptor+autoantibodies+in+patients+with+first-episode+psychosis+from+the+OPTiMiSE+project.">Cell- and single molecule-based methods to detect anti-n-methyl-d-aspartate receptor autoantibodies in patients with first-episode psychosis from the OPTiMiSE project.</a> Biol Psychiatry. , (2017).</li></ol>

저자는 공개 없다.

라이브 세포 기반 면역 형광 분석 결과11, 고정 세포 기반 면역 형광 분석 결과9 포함 하 여 문학에서 보고 된 NMDA 수용 체에 대 한 신경의 존재를 화면에 여러 세포 기반 면역 분석을 있다 , cytometry 기반 흐름 시험14및. 라이브 세포 기반 면역 형광 분석 결과 흐름 cytometry 기반 분석 결과 훈련 된 인력과 특수 장비를 요구 하면서 ectopically NR1 단백?...

안티 NMDA 수용 체 면역 뇌염 새로 인식 된 질병 엔터티는 모든 연령대의 환자에서 발생할 수 있으며 주로 여성 환자1,2에 영향을 미치는입니다. 그것은 가장 자주 진단된 뇌염 뇌염3의 초기 알 수 없는 병을 가진 환자 들 중 하나입니다. 일반적으로 안티-NMDA 수용 체 뇌염으로 영향을 받는 환자는 두통이 나 발열, 의식 수준의 변화 및 다양 한 동요, 과민성을 포함 한 급성 정신병 증상의 빠른 발전에 의해 다음의 prodromal 증상이 불안, 불면증, 환각, 망상, 침략, 기괴 한 행동, 운동 이상, 자율 dysregulation, 그리고 발작 공격4,5. 이 조건과 immunotherapy 적시 치료의 초기 인식 더 나은 결과 영향을 받는 환자6전체 복구에 대 한 중요 하다. 따라서, 안티-NMDA 수용 체 면역 뇌염 환자 급성 또는 새로운 발병 정신병 특징7,8제시의 중요 한 감 별 진단으로 고려 되어야 한다 건의 된다.
임상 기능 외 autoantibody CSF 또는 혈액에 NMDA 수용 체에 대 한의 안티 NMDA 수용 체 면역 뇌염9의 정확한 진단을 위해 필수적 이다. 안티-NMDA 수용 체 autoantibody 감지 하 면역 테스트의 대부분은 몇 가지 연구 실험실10,의11, 사용할 수 거기 이며 심사에 대 한 단 하나의 상용 세포 기반 면역 형광 분석 결과 안티-NMDA 수용 체 신경12. 이 연구의 목표는 사용할 수 있는 편리 하 게 실험실에서 안티 NMDA 수용 체의 임상 연구를 촉진 하기 위하여 안티 NMDA 수용 체 신경의 존재를 화면에 간단한 사내 세포 기반 면역 형광 분석 결과 개발 하는 뇌염은 면역 NMDA 수용 체는 heterotetramer 이온 채널 단백질 복잡 한 두뇌에서 구체적으로 표현. 그것은 두 개의 필수 NR1 소 단위, 및 NR2A, NR2B, NR2C, 또는 NR2D13의 하나 또는 두 개의 소 단위 조합의 만들어진다. 이전 연구는 항 체에 의해 대상 주요 epitope NR1 소 단위5의 extracellular N 맨끝 도메인에는 보고. 따라서,이 프로토콜에 우리 인간 미 발달 신장 상피 세포 라인 (HEK293), 녹색 형광 단백질 (GFP) 태그로 NMDA 수용 체의 인간 재조합 형 NR1 소 단위 단백질을 표현 하 고 개발 감지 하는 세포 기반 면역 형광 분석 결과 안티-NMDA 수용 체 신경에 혈액의 IgG 클래스.

<table>
	<tbody>
		<tr>
			<td>GRIN1 (GFP-tagged) - Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1</td>
			<td>Origene (Rockville, MD, USA)</td>
			<td>RG219368</td>
			<td>NR1-cDNA clone tagged with C-terminal tGFP sequences</td>
		</tr>
		<tr>
			<td>pCMV6-AC-GFP</td>
			<td>Origene (Rockville, MD, USA)</td>
			<td>PS100010</td>
			<td>mammalian vector with C-terminal tGFP tag</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen (Hilden Germany)</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen (Carlsbad, CA, USA)</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, Pyruvate</td>
			<td>Thermo Fisher</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Characterized Fetal Bovine Serum, US Origin</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td>Thermo Fisher</td>
			<td>A-11014</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive control: anti-glutamate receptor (type NMDA)</td>
			<td>Euroimmun AG, L&#252;beck, Germany</td>
			<td>CA 112d-0101</td>
			<td></td>
		</tr>
		<tr>
			<td>Euroimmun Assay Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple cell-based immunofluorescence assay to detect autoantibody against the n-methyl-d-aspartate (nmda) receptor in blood

안티-NMDA 수용 체 autoantibody의 존재 안티 NMDA 수용 체 면역 뇌염 되 나 영향을 받는 환자에서 다양 한 정신병 증상을 일으킬 수 있습니다. 혈액 또는 뇌 척추 액체 (CSF)에 NMDA 수용 체에 대 한 특정 autoantibody의 검출은이 조건의 정확한 진단을 위해 필수적 이다. NMDA 수용 체는 이온 채널 단백질 복잡 한 두 필수 NMDA 수용 체 소 단위 1 (NR1)와 하나 또는 두 개의 NMDA 수용 체를 포함 하 여 4 개의 소 단위를 포함 하는 소 단위 2A (NR2A), NMDA 수용 체 소 단위 2B (NR2B), NMDA 수용 체 소 단위 2 C (NR2C), 또는 NMDA 수용 체 소 단위 2D (NR2D). 안티-NMDA 수용 체 autoantibody epitope NMDA 수용 체의 NR1 소 단위의 extracellular N 맨끝 도메인에 있어야 보고 되었다. 이 연구의 목표는 NMDA 수용 체의 NR1 소 단위에 대 한 신경의 임상 및 기초 연구를 촉진 하기 위하여 혈액에서 검출 하는 검사로 사용할 수 있는 간단한 세포 기반 면역 형광 분석 결과 개발 하는 안티-NMDA 수용 체 면역 뇌염

평균적으로, 우리는 프로토콜의 섹션 1에서에서 설명한 절차에 따라 250 mL 세균성 문화에서 200-300 µ g 내 무료 식 플라스 미드 NR1 GFP와 GFP를 얻을 수 있습니다. 프로토콜의 섹션 2에에서 설명 된 대로 48-잘 접시에 배양 HEK293 세포의 transfection는 양의 식 플라스 미드의 100 ng/잘 사용 되었다. transfection 후 24-30 h, 셀 표현 NR1 GFP 그리고 GFP 재조합 단백질 형광 현미경에서 검출 될 ?...

Watch this Scientific Journal Video about A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood at JoVE.com

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

우리는 ectopically NMDA 수용 체에 대 한 자가 면역 뇌염으로 의심 되는 환자의 혈액에서 검출 하는 항으로 인간 배아 세포 (HEK293)에서 녹색 형광 단백질 태그로 NMDA 수용 체의 NR1 소 단위를 표현. 이 간단한 방법 임상 설정에서 심사 적합 있을 수 있습니다.

Autoantibody N-메 틸-D-Aspartate (NMDA) 수용 체에 대 한 혈액에서 검출 하는 간단한 세포 기반 면역 형광 분석 결과

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

a-simple-cell-based-immunofluorescence-assay-to-detect-autoantibody

Research

JoVE Journal

Neuroscience

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

9.8K 조회수.  Chang Gung Memorial Hospital-Linkou. 우리는 ectopically NMDA 수용 체에 대 한 자가 면역 뇌염으로 의심 되는 환자의 혈액에서 검출 하는 항으로 인간 배아 세포 (HEK293)에서 녹색 형광 단백질 태그로 NMDA 수용 체의 NR1 소 단위를 표현. 이 간단한 방법 임상 설정에서 심사 적합 있을 수 있습니다.

비디오: A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

동적 클램프 기법을 사용 랫 Midbrain Dopaminergic 뉴런에서 NMDA 수용체의 전도의 응용

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

연구

신경과학

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

파리에서 에탄올 민감도를 측정하는 간단한 방법

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

이미징은 기본 양식 마우스 뉴런의 플라즈마 막에 수용체 삽입을 pHluorin 태그

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

테스트 시각 기능에 대한 간단한 행동 분석<em&gt;는 Xenopus laevis의</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

마우스에 콜드 민감도 및 적응을 결정하기위한 간단하고 저렴한 방법

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

사용 보상 추구 행동의 변화를 측정하는 간단한 방법<em&gt; 노랑 초파리</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

기능과 Neuropathological 평가 대 한 C57BL/6 쥐에 실험 자가 면역 신경 염을 유발 하는 간단한 방법

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

탐지 기술 현장에서 혁신적인 RNA에 의해 설치류 핵 Accumbens에서 도파민 D1-알파 수용 체의 식별

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

글리신/D-떠들고와 조미료에 감도와 NMDA 수용 체를 공부 하는 높은 처리량 칼슘-플럭스 분석 결과

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...