우리는 시설과 이온 채널의 연구에 대한 훌륭한 환경으로 우리를 제공 생리학 및 분리막 생물, 캘리포니아 데이비스 대학의학과에 감사하고 있습니다.

1. 휴식 인간의 T Lymphocytes의 준비 <ol><li> RosetteSep 인간 T 세포 농축 칵테일과 RosetteSep 밀도 매체를 사용하여, 제조 업체의 지시에 따라 인간의 혈액 샘플에서 T 세포를 정화. 그 결과 세포 인구는 95 % 인 경우에는 3 번 CD + 쉬고있는 T 세포를 포함해야합니다. 우리는 UC 데이비스 내부 검토위원회의 승인을 프로토콜에 따라 건강한 지원자에서 수집한 말초 혈액 샘플에서 인간의 T lymphocytes 정화. </li><li> 분리 후 10 % 태아 송아지 혈청, 2퍼센트 GlutaMAX - I, 1 % RPMI 1640과 보충, 글루타민과 HEPES와 RPMI - 1640 매체를 포함하는 세포 배양 매체에서 0.5 X 10 6 세포 / ML의 밀도에 쉬고 T 세포를 resuspend 비타민 용액, 1 % RPMI 1640 아미노산 용액, 1 %의 나트륨 pyruvate, 그리고 0.03 % β - 메르 캅 토 에탄올. </li><li> 플레이스 세포 세포 배양 flasks에 서스펜션과 ° humidified 인큐베이터에서 C 5 % CO 2와 함께 최대 24 H하기 전에 실험을 위해 37 유지합니다. </li></ol> 2. 기록 회의소의 준비 <ol><li> 다음은 녹화 회의소를 준비하는 데 필요한 :) B, A) 실리콘 O - 링을 회전, 70 % 에탄올과 증류수 H 2 O, C와 함께 청소를 25 mm coverslips)는 Sylgard184를 혼합 제조 업체의 지시에 따라 준비하고, D) 이 60 X 15mm 페트리 요리. </li><li> 플레이스 ~ 60x15 - mm 배양 접시에 혼합 Sylgard184 0.5 ML. </li><li> Sylgard184 사용 포셉에 O - 링의 하단 부분을 찍어. </li><li> 같은 다른 배양 접시로 깨끗한 표면에 O - 링을 배치하여 O - 링의 가장자리에서 초과 Sylgard184를 제거합니다. </li><li> coverslip에 반지를 놓고 그것을 아래로 누르십시오. </li><li> 85 ° 40-60 분 또는 Sylgard184까지 C가 polymerized입니다에서 실 가열하여 Sylgard184 치료. </li><li> 먼지없는 용기에 녹화 실에 보관하십시오. </li></ol> 3. 패치 Pipettes의 준비 <ol><li> 실험 당일, 유리 모세관 및 피펫 풀러를 사용하여 ~ 2 μm의 팁 직경 pipettes를 가져옵니다. 우리는 필라멘트 (1.5 mm 및 ID : 1.10 mm OD)와 borosilicate 튜빙을 사용합니다. 먼지없는 위치에 저장 pipettes. </li><li> 표준 절차에 따라 9 Sylgard184 또는 HIPEC R6101 반도체 보호용 코팅 코트 피펫은 팁. </li><li> 부드럽게 실험 전에 pipettes를 해고 - 폴란드어. </li></ol> 4. 패치 클램프 설치 준비 <ol><li> 여러분의 패치 - 클램프 앰프를 제어 소프트웨어의 gigaseal 형성에 대한 매크로를 구성하고 저장합니다. 우리는 Windows XP 및 데이터 수집을위한 펄스 소프트웨어에 의해 지원되는 EPC - 10 앰프를 사용합니다. 우리는 EPC - 10 사용 설명서에 따르면, &quot;에 셀 &#39;및&#39;전체 셀&quot;매크로 &quot;설정&quot;을 설정하고 모든 실험에 사용합니다. </li><li> 이전 실험 목욕 솔루션 및 표 1에 나와있는 피펫 솔루션을 준비하고 최대 일주 4 ° C에 그들을 저장할 수 있습니다. </li></ol><table border="1"><tbody><tr><td> 화학 제품 </td><td colspan="3"> 바스 솔루션 (㎜) </td><td> 피펫 솔루션 (㎜) </td></tr><tr><td></td><td> # 1 </td><td> # 2 </td><td> # 3 </td><td></td></tr></tbody><tbody><tr><td> CH 3 SO 3 나 </td><td> 130 </td><td> 110 </td><td> 125 </td><td> - </td></tr><tr><td> NaCl </td><td> 2 </td><td> 4 </td><td> 5 </td><td> - </td></tr><tr><td> CA (OH) 2 </td><td> - </td><td> 20 </td><td> - </td><td> - </td></tr><tr><td> MgCl 2 </td><td> 3 </td><td> 1 </td><td> - </td><td> 5 </td></tr><tr><td> MgSO 4 </td><td> - </td><td> - </td><td> - </td><td> 2 </td></tr><tr><td> HEPES </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 15 </td></tr><tr><td> HEDTA </td><td> - </td><td></td><td> 10 </td><td> - </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> - </td><td> - </td><td> 1 </td><td> - </td></tr><tr><td> CS -spartate </td><td> - </td><td> - </td><td> - </td><td> 125 </td></tr><tr><td> BAPTA </td><td> - </td><td> - </td><td> - </td><td> 12 </td></tr><tr><td> 포도당 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> - </td></tr></tbody></table> 표 1. 전체 셀 CARC 현재 레코딩을위한 솔루션을 제공합니다. 모든 화학 물질은 시그마 - 알드리치, 세인트 루이스, MO에서 구입한했다. <ol start="3"><li> 패치 피펫 솔루션과 각 목욕 솔루션 10 ~ 액상 접합 잠재력 (LJ)를 결정합니다. 당신의 수집 소프트웨어를 사용하여 적절한 장소에 LJ 값을 저장합니다. 예를 들어, 펄스 프로그램의 &quot;앰프&quot;창에서 &quot;LJ&quot;컨트롤에 LJ 값을 삽입합니다. </li><li> 실험 전에 수집 소프트웨어의 적절한 장소에서 그림 1에 표시된 자극 프로토콜, 구성 및 저장합니다. 펄스 프로그램에서 우리는 &quot;펄스 생성&quot;창에서 수집 프로토콜을 설정합니다. <img src="/files/ftp_upload/2346/2364fig1.jpg" alt="그림 1" /> 그림 1. . 자극 프로토콜 -120에서 기간 50 MS의 백 뮤직 비디오에 전압 램프마다 0.2를 적용 - 2 s을 (5-0.5 Hz에서). 구불 구 사이의 개최 가능성 (V H)는 30 MV에서 관리하고 있습니다. </li></ol> 5. 현미경 스테이지에 장착 셀 <ol><li> 세포를 도금하기 전에 다음 ~ 20 분 및 세척 H 2 O와 행크의 균형 소금 용액 (HBSS)에 대한 폴리 - L - 라이신과 코트 녹화 실 있습니다. </li><li> 37 20 분 챔버 및 부화에 0.2-0.5 X10 6 셀을 포함하는 세포 현탁액의 플레이트 500 μL ° C. </li><li> 챔버 홀더에 부착된 세포로 녹음 챔버를 고정합니다. 우리는 맞춤 제작 챔버베이스 coverslip의 바닥을 지원 홀더, coverslip의 상단으로부터 상단 부분은 부드럽게 프레스 동안.를 사용하여 </li><li> 거꾸로 현미경의 무대에 챔버 홀더를 놓습니다. </li><li> 전송 빛의 세포에 초점. 우리는 40X 기름 침지 목표를 사용합니다. </li><li> 욕조에 자세 / AgCl 기준 전극을 삽입합니다. </li><li> multibarrel 중력 기반의 재관류 시스템을 맞춥니다. 유입 튜브의 끝은보기의 필드에 coverslip 가까이에 45 ° 각도로 배치해야합니다. </li><li> 유입 튜브 끝에 반대 흡입 튜브를 놓습니다. </li><li> 와 챔버 Perfuse 인산염 버퍼 호수 (PBS) 또는 HBSS은 세포 배양 매체와 느슨하게 연결된 세포를 멀리 세척하십시오. </li></ol> 6. CRAC 채널 해류 녹음 <ol><li> 단단히 coverslip에 연결된 세포를 찾아보십시오. 우리는 일반적으로 부드러운 외부 표면과 평균 크기, 원형 셀을 선택합니다. </li><li> + - 무료, 3 MM MG 2 + 함유 목욕 솔루션 SERCA 펌프를 차단하기 위해 80-10 분 0.5-1 μm의의 thapsigargin를 포함하는 # 1 (표 1) 칼슘 2 녹음 챔버를 Perfuse. 우리는 이전 gigaseal 형성하기 위해 저장소를 고갈이 단계를 수행합니다. </li><li> Eppendorf microloader와 P - 20 변위 피펫을 사용하여, 피펫 솔루션 (표 1)과 함께이 패치 피펫을 기입하여 증폭기 headstage에 피펫 홀더에 패치 피펫를 삽입합니다. 칼슘 2 + chelator BAPTA는 수동적으로 저장을 고갈하고 + 종속 CRAC 채널 불활 성화 칼슘 2 줄이기 위해 피펫 솔루션에 포함되어 있습니다. </li><li> 패치 피펫 내부 욕조에 들어가기 전에 긍정적인 압력 (H 2 O 중 ~ 3 인치)의 작은 금액을 적용합니다. 우리는 가압 공기와 패치 피펫 내부 긍정적이거나 부정적인 압력을 만들 진공 라인에 연결된 사용자 정의 - 만든 압력 흡입 장치를 사용합니다. </li><li> 현미경을 통해 시각적인 통제 욕조에 패치 피펫을 낮추십시오. &quot;오실로 스코프&quot;창에서, 당신은 피펫을 통해 흐르는 전류를 볼 수 있습니다. 현재 진폭의 단계와 같은 변경 사항이 미리 매크로에 의해 적용되는 작은 진폭 전압 펄스 (시험 펄스)에 의해 유도해야합니다. 이 시점에서, 당신은 피펫 저항의 가치를 확인할 수 있어야합니다. 우리 pipettes의 저항은 일반적으로 5-6 MΩ입니다. </li><li> 피펫 및 참조 전극 사이에 생성된 &quot;오프셋&quot;잠재력을 수정합니다. </li><li> 그것은 세포막을 만지는 때까지 현미경을 통해 시각적인 통제, 가까이 세포에 피펫 팁을 가지고. </li><li> 패치 피펫 내부 부정적인 압력 (H 2 O의 20~40인치)을 적용합니다. </li><li> &quot;오실로 스코프&quot;창에서 gigaseal 형성을 모니터링합니다. 당신은 시험 펄스, 감소 및&gt; 5 GΩ에 피펫 저항 증가의 가치에 의해 유도 전류의 진폭을 볼 수 있습니다. 철 이내 gigaseal 형태w 초,하지만 그것은 안정 gigaseal을 달성하기 1-2 분 정도 걸릴 수 있습니다. </li><li> 피펫 커패시턴스 ( &quot;C - 빠른&quot;)에 대한 보상. </li><li> 20 CC 주사기를 사용하여 추가 부정적인 압력을 적용하여 패치 막 브레이크. </li><li> 30 MV에 보관 잠재력을 설정합니다. 긍정적인 지주 잠재 칼슘 의존 CRAC 채널 불활 성화를 방지하는 데 도움이됩니다. </li><li> 세포의 막 커패시턴스 ( &quot;C - 느린&quot;)에 대한 보상, C - 느린의 가치를 저장하거나 기록합니다. </li><li> 액세스 저항 &quot;R S&quot;의 가치를 확인합니다. 우리의 실험에서는 R S는 일반적으로 MΩ 70-20 있습니다. </li><li> + 무료 3 MM MG 2 + 함유 목욕 솔루션 # 1 칼슘 2 0.5 Hz의 주파수 (그림 1)과 전압 구불 구 일련의 사항을 적용하려면 시작합니다. MG 2 +에 대한 CRAC 채널의 가난한 투자율에 의한 &quot;누설&quot;현재와 비교하여 본 솔루션에서는 CRAC의 전류 negligibly 작습니다. 저장하고 &quot;누출&quot;조류로서 향후 분석에서 목욕 솔루션 # 1에 기록된 현재 추적을 사용합니다. -100 MV에있는 &quot;누설&quot;현재의 절대 진폭 미만 또는 5 PA 동일해야합니다. 세포는 &quot;새는&quot;있다면, 새로운 패치 피펫하고 새로운 세포를 사용을 통해 실험 시작을 중지합니다. </li><li> CRAC 채널 (I CA - CRAC)를 통해 칼슘 2 + 전류를 기록하려면 재관류 밸브 전환 20 MM 칼슘 2 + 함유 목욕 솔루션 # 2 (표 1)과 칼슘 2 + 무료 3 MM MG 2 + 함유 목욕 솔루션 # 1을 대체합니다. 이것은 칼슘 2 + CRAC 채널을 통해 흐름에 가능하며 내향 전류를 생산하고 있습니다. &quot;오실로 스코프&quot;창에서, 당신은 내심 I CA - CRAC (그림 2) 정류의 개발을 볼 수 있습니다. 부정적인 전압에서 전류 진폭은 칼슘 2 CRAC 전류의 + 종속 potentiation로 인해 약 1 분 계속 증가할 것입니다. </li><li> CRAC 채널을 통해 과도 나 + 전류를 기록하는 데 필요한 5 Hz에서, 전압 경사로와 함께 자극의 빈도를 늘리십시오. </li><li> 20 MM를 교체 칼슘 2 + 함유 재관류 밸브를 전환하여 나 + 함유 이가의 양이온 무료 솔루션 # 3 (표 1) 욕조 솔루션 # 2. &quot;오실로 스코프&quot;창에서, 당신은 안쪽으로 나를 정류 큰 진폭의 과도 발전을 볼 수 + ​​현재 CRAC 채널 (I 나 - CRAC, 그림 2)를 통해. </li><li> 하나는 20 MM 칼슘 2 &quot;누설&quot;현재를 기록하는 실험의 끝에 1-5 μm의 라 3 +를 포함하는 + 함유 목욕 솔루션 # 2를 적용할 수 있습니다. 그러나, 우리는 시간이 지남에 &quot;누출&quot;현재 변경하기 때문에이 방법은 유용하지 않았고 그것은 실험의 끝에 크거나 작게 될 수 있습니다. 우리는 칼슘이에 기록된 현재 + 무료 3 MM MG 2 &quot;누설&quot;현재와 같은 실험의 시작 부분에 + 함유 목욕 솔루션 # 1.을하는 것을 선호 </li><li> 자세한 분석을 위해 기록된 현재의 흔적을 저장합니다. </li></ol> 7. 데이터 분석 <ol><li> 분석 소프트웨어에 저장된 현재 레코드를 검색할 수 있습니다. 우리는 분석을위한 펄스 프로그램을 사용합니다. </li><li> 처음에과 전압 경사로의 끝부분에 현재 amplitudes를 측정. 우리는 일반적으로 -100 뮤직 비디오에서와 펄스 프로그램의 &quot;오실로 스코프&quot;창에서 커서의 쌍을 사용하여 100 뮤직 비디오에서 현재 amplitudes를 측정합니다. </li><li> 자세한 분석 및 그래픽 프레 젠 테이션 그래픽 프로그램으로 현재 amplitudes의 가치를 내보냅니다. 우리는 기원 과학 그래프 및 분석 소프트웨어, 버전 7을 사용합니다. </li></ol> 8. 대표 결과 <img src="/files/ftp_upload/2346/2346fig2.jpg" alt="그림 2" /> 그림 2. 쉬고 인간 T 세포의 CRAC 전류. (A), -100 MV (채워진 원)과 100 MV (오픈 원)에서 전체 셀 전압 클램프 구성에 기록된 CRAC 전류의 시간 코스. gigaseal 형성하기 전에, 세포는 칼슘 2 ~ 10 분 preincubated했습니다 + 무료 3 MM MG 2 + 함유 목욕 솔루션 0.5 μm의의 thapsigargin을 포함하는 # 1. 침입 후, 목욕 솔루션은 순차적으로 다음과 같이 적용되었습니다 칼슘 2 + 무료 3 MM MG 2 칼슘 2 + 함유 목욕 솔루션 # 2 20 MM 다음 + 함유 목욕 솔루션 # 1 (0 칼슘 + 3 MG) (20 CA), 목욕 솔루션 # 2 (20 CA) 뒤에 이가의 양이온 무료 목욕 솔루션 # 3 (DVF)로 따라갔다. 전지는 그림 1에 표시된 전압 구불 구 일련의 자극했다. 구불 구의 빈도는 목욕 솔루션 # 3 (DVF)와 다른 모든 솔루션에 0.5 Hz에서 5 Hz에서했습니다. 20 MM 칼슘 2 + 함유 목욕 솔루션 # 2 및 DVF 목욕 솔루션 # 3 I 나 - CRAC의 빠른 과도 개발 신청 후 CA - CRAC의 느린 개발을합니다. (B), 대표 현재 흔적이 + 무료 3 MM MG 2 + 함유 목욕 솔루션 # 1 (CA 2 전압 경사로 동안 기록&quot;누설&quot;), 20 MM 칼슘 2 + 함유 목욕 솔루션 # 2 (20 CA), 그리고 목욕 솔루션 # 3 (DVF)을. (C, D), I CA - CRAC (C)와 나 - CRAC (D)의 전류 - 전압 관계 20 MM의 전압 - 경사로 중에 칼슘 2 + 함유 목욕을 기록 전류에서 &quot;누출&quot;현재를 빼서 얻은 솔루션 # 2 (20 CA), 그리고 목욕 솔루션 패널에 표시된 # 3 (DVF) (B).

<ol>
	<li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRAC+channelopathies.">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li><li>Lewis, R. S., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+inactivation+of+depletion-activated+calcium+current+(ICRAC)+due+to+local+calcium+feedback.">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms.">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes.">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2+-inhibited+cation+(MIC)+channels.">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li><li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+channel+properties+and+regulated+expression+of+Ca(2+)+release-activated+Ca(2+)+(CRAC)+channels+in+human+T+cells.">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+for+liquid+junction+potentials+in+patch+clamp+experiments.">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이러한 세포의 내생 CRAC 현재 진폭이 작은 셀 크기 (쉬고 인간 T 세포의 직경은 5-8 μm의 범위에있는)에 의한 소형이기 때문에 인간의 T 세포를 휴식에 CRAC 전류 electrophysiological 조사 어려운 작업입니다. 여기에서 우리는 확실하게 주변 혈액 mononuclear 세포로부터 격리 쉬고있는 인간의 T lymphocytes에 CRAC 전류를 기록하기위한 단계별 절차를 제시한다. 이 기술은 우리가 더 나은 정상 및 병적인 면역 세?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RosetteSep Human T Cell Enrichment Cocktail</td>
<td>&nbsp;</td>
<td>StemCell Technologies, Vancouver, BC, Canada</td>
<td>15061</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep Density Medium</td>
<td>&nbsp;</td>
<td>StemCell Technologies </td>
<td>15705</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-in 1640 medium w/glutamine/HEPES</td>
<td>&nbsp;</td>
<td>Fisher, Waltham, MA</td>
<td>SH3025501</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum</td>
<td>&nbsp;</td>
<td>Omega Scientific, Tarzana, CA</td>
<td>FB-01</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX-I (100X solution)</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 vitamin solution (100X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>7256</td>
<td>&nbsp;</td>
<tr>
<td>1640 amino acids solution (50X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R7131</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Inositol trisphosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>19766</td>
<td>&nbsp;</td>
<tr>
<td>BAPTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A4926</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Lanthanum Chloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>262072</td>
<td>&nbsp;</td>
<tr>
<td>Thapsigargin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>586005</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
<tr>
<td>HIPEC R6101 Semiconductor Protective Coating</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>63-500 Series High-Performance Vibration Isolation Lab Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing, Peabody, MA</td>
<td>63-540</td>
<td>&nbsp;</td>
<tr>
<td>EPC 10 patch clamp amplifier with headstage</td>
<td>&nbsp;</td>
<td>HEKA Instruments, Bellmore, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator </td>
<td>&nbsp;</td>
<td>Sutter Instrument, Novato, CA </td>
<td>MP-285</td>
<td>&nbsp;</td>
<tr>
<td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td>
<td>&nbsp;</td>
<td>Olympus America, Center Valley, PA</td>
<td>1X71</td>
<td>&nbsp;</td>
<tr>
<td>Windows Computer</td>
<td>&nbsp;</td>
<td>Dell </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pulse software</td>
<td>&nbsp;</td>
<td>HEKA Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Origin Scientific Graphing and Analysis Software</td>
<td>&nbsp;</td>
<td>OriginLab, Northampton, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Patch pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF150-110-7.5</td>
<td>&nbsp;</td>
<tr>
<td>Narashige's Microforge</td>
<td>&nbsp;</td>
<td>Tritech Research, Los Angeles, CA</td>
<td>MF-830</td>
<td>&nbsp;</td>
<tr>
<td>Silicon O-rings </td>
<td>&nbsp;</td>
<td>McMASTER-CARR, Santa Fe Springs, CA</td>
<td>111 S70</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips 25 mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>12-545-102 25 mm 25CIR.-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

T lymphocytes 년, CA 2 고갈 + 세포내 칼슘 2 + 저장소, plasmalemmal 칼슘 2 활성화에 + 채널을 리드 칼슘 출시가 활성화된 칼슘 (CRAC) 채널을했다. CRAC 채널은 T 세포 증식 및 유전자 발현의 조절에 중요​​한 역할을한다. T 세포의 비정상적인 CRAC 채널 기능이 심한 결합 면역 결핍과 면역 질환 1, 2에 연결되어 있습니다. 인간 T 세포의 CRAC 채널 기능을 공부하는 것은 정상적인 면역 반응을 조절 새로운 분자 메커니즘을 발견하고 관련 인간 질병의 원인을 풀다 수 있습니다. 멤브레인 전류의 Electrophysiological 녹음 기능 채널 특성과 규제의 가장 정확한 평가를 제공합니다. Jurkat T 세포, 인간의 백혈병 T 세포 라인에 CRAC 채널 전류의 Electrophysiological 평가는 처음 3 전 20 년 이상 수행했다, 그러나 정상적인 인간의 T 세포에 CRAC 전류 측정이 어려운 과제 남아있다. 정상적인 T 세포의 CRAC 채널 전류를 기록의 어려움은 혈액 파생 T의 lymphocytes가 Jurkat T 세포보다 크기가 훨씬 작고, 따라서 내생 전체 세포 CRAC 전류가 진폭 매우 낮게있다는 사실에 의해 혼합 수 있습니다. 여기, 우리가 정기적으로 칼슘 2를 기록하는 데 사용하는 + 나 + 나 건강한 자원 봉사자의 말초 혈액에서 분리된 인간의 T 세포를 휴식에 CRAC 채널을 통해 전류. 단계별 절차를 제공 방법은 여기에 설명된 Jurkat T 세포에서 CRAC 전류를 기록하고 인간의 T 세포 4-8을 활성화에 사용되는 절차에서 채택되었습니다.

Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

우리는 칼슘 출시가 활성화된 칼슘의 전체 세포 패치 클램프 녹음 (CRAC) 말초 혈액 mononuclear 세포 - 파생 인간 T의 lymphocytes의 전류에 대한 단계별 절차를 제공합니다.

인간의 T Lymphocytes에서 칼슘 출시가 활성화된 칼슘의 전체 세포 기록 (CRAC) 해류

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

whole-cell-recording-calcium-release-activated-calcium-crac-currents

Research

JoVE Journal

Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

16.0K 조회수.  University of California, Davis. 우리는 칼슘 출시가 활성화된 칼슘의 전체 세포 패치 클램프 녹음 (CRAC) 말초 혈액 mononuclear 세포 - 파생 인간 T의 lymphocytes의 전류에 대한 단계별 절차를 제공합니다.

비디오: Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

칼슘 지표로 로딩 Drosophila 신경 터미널

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

연구

생물학

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

레이어의 VIVO에서 2 광자 칼슘 이미징 마우스의 2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

라이트의 전체 세포 레코딩은 망막 통에 흥분성의 시냅틱 전류를 Evoked

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Astrocytes의 플라즈마 막 글로벌 세포내 칼슘 역학 니어 측정

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes의 빠른 칼슘 Fluxes 측정

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

실시간 Vivo에서 녹음 애기 칼슘 신호의 곤충 먹이 형광 바이오 센서를 사용 하 여 중

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

마우스 췌도 세포의 하위 집단에 있는 화상 진찰 칼슘 역학

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...