비전 건강 연구 네트워크 -이 작품은 건강 연구 (178918), 퐁 드 공들인 엉 상테 퀘벡의 캐나다 연구소에서 보조금에 의해 지원됩니다.

참고 : 남성 C57BL / 6 마​​우스 (5-7주 이전)의 찰스 리버 연구소 인터내셔널 (. 세인트 - 상수, 퀘벡, 캐나다)에서하고 CHU 셍트 - 저스틴 동물 관리위원회에 의해 승인 된 프로토콜에 따라 조작 할 수 있습니다. 마우스 기관지 조직 절편은 상피 세포에 LMPs의 아폽토시스 효과를 조사하는 차 기관지 상피 세포의 좋은 소스를 제공합니다. 이 프로토콜은 LMPs의 시험 관내 생성뿐만 아니라 LMPs 처리 기관지 조직 이식편에 대한 상피 세포 자멸사를 검출하기위한 방법을 설명한다. 이 프로토콜은 세 부분으로 구성되어 있습니다. 1. LMPs 생산 및 특성 참고 : 오염을 방지이 실험에 사용 된 모든 재료는 멸균 또는 멸균되어 있는지 확인하십시오. 달리 언급하지 않는 한, 무균 조건에서 생물 안전 캐비닛에 실온에서 모든 단계를 수행합니다. 1.1) 자극과 의원의 컬렉션9 <ol><li> 37 ° C의 수욕 천만 CEM T 세포의 분취 량을 해동. 10ml에 희석 예열 된 무 혈청 200 GX 5 분에서 15 ㎖의 멸균 튜브와 원심 분리기, 예컨대 X-VIVO 같은 조혈 매체. 예열 배지 5ml에 재현 탁하고 세포 상등액을 기음. </li><li> 15 ml의 예열 조혈 같은 X-VIVO와 같은 매체 5 % CO 2와 37 ° C에서 가습 배양기에서 4 일 동안 배양과 (서스펜션 세포) T75 조직 배양 플라스크에 넣고 세포를 전송합니다. </li><li> 사일 후, 100 ㎖의 새로운 배지를 함유하는 T175 조직 배양 플라스크에 모든 배양액과 세포를 옮긴다. 그들은 2 백만 세포 / ml의 밀도로 성장 될 때까지 동일한 조건에서 약 72 시간 동안 계속하여 배양 된 세포. </li><li> 균등 네 T175 플라스크 각각 포함 된 150 ml의 신선한 매체 세포가 200 만 / ml의 농도로 (약 48 시간 배양)를 재배 할 때까지 세포 배양을 계속 사이에 세포를 분리. </li><li&gt; 5 분 동안 200 XG에서 원심 분리하여 각 플라스크로부터 세포를 수집하고, 2 백만 / ml의 세포 밀도를 유지하기 위하여, 150 ㎖의 새로운 배지를 함유하는 새로운 T175 플라스크에 300 × 106 세포를 재현 탁. </li><li> 0.5 ㎍ / ml의 최종 농도로 배지 (2 ㎎ / ㎖로 DMSO에 용해) 티노 마이신 D 첨가하고 24 시간 동안 배양한다. </li><li> 50 ML 원뿔 튜브에 모든 문화 매체를 전송하고 5 분 동안 750 XG에서 세포를 스핀 다운. 15 분 대 세포 파편을 제거하기 위해 1,500 XG에 50 ML 원뿔 튜브와 원심 분리기에 뜨는을 전송합니다. </li><li> 50 분 동안 12,000 XG에 250 ml의 병 및 초 원심 분리기에 뜨는을 전송합니다. 뜨는을 취소하고 알약을 수집합니다. </li><li> 워시 50 분 동안 12,000 XG에서 원심 분리하여 50 ㎖ 튜브에 40ml의 무균 PBS로 펠릿 LMPs이 농후. 두 번이 단계를 반복합니다. </li><li> 마지막 세척 상층 액을 수집; 이는 비히클 대조군으로 사용한다. 1 LMPs 펠렛을 일시 중단㎖의 PBS는 1.5 ml의 멸균 마이크로 튜브로 전송할 수 있습니다. 분취 매장 절연 LMPs는 -80 ° C에서 (다중 자유 - 해동 사이클을 방지하기 위해). </li></ol> FACS 분석 (4)를 통해 의원 1.2) 특성 <ol><li> 염화칼슘이없이 넥신 버퍼의 두 샘플 1과 다른 준비 : 헤 페스 10 mM의 NaCl을 140 mM의 플러스 마이너스 5 mM의 염화칼슘 2. </li><li> 입자를 제거하기 위해 0.22 μm의 필터를 사용하여 아 넥신 FACS 완충액 시스 유동 유체 필터. </li><li> FACS 튜브에 5 mM의 염화칼슘 2 넥신 버퍼의 44 μL에 LMPs 1 μl를 희석. 염화칼슘 2 (음성 대조군)없이 넥신 버퍼의 44 μL에 LMPs 1 μL와 다른 튜브를 준비합니다. </li><li> 각각의 튜브에 넥신 - Cy5에 5 μl를 넣고 잘 섞는다. 어둠 속에서 실온에서 15 분 동안 품어. 각 튜브에서 FACS 시스 유동 유체의 400 μL와 혼합하여 희석함으로써 반응을 정지. </li><li> 7 μm의 계수 BEA의 10 μL (20 구슬)를 추가합니다각 튜브에서 내부 표준으로서 DS 현탁액 절대 수를 얻었다. </li><li> 상대적 크기의 게이트 (FSC-H, PMT E00, 규모 로그인) 및 상대 단위 (SSC-H, PMT (325), 로그 스케일)의 크기 보정 형광 구슬을 사용하여 흐름 cytometer에 도트 플롯을 설정 1 ㎛ (게이트 1) 7 μm의 (문 2)에서 비즈 게이트를 계산. </li><li> 20,000 카운트 비즈 게이트 2에 도달 할 때까지 신호를 획득함으로써, 도트 플롯 (규모를 기록, PMT 765) 넥신에 대한 설립 게이트 및 FL-4 채널을 사용하여 FSC-H / SSC-H 플롯에 LMPs 샘플을 분석 할 수 있습니다. </li><li> CaCl2를 포함 넥신 버퍼에 LMPs의 긍정적 인 넥신 이벤트를 결정하고 염화칼슘 2 (음성 대조군)없이 넥신 버퍼에 LMPs의 이벤트를 뺍니다. </li><li> 다음의 식에 기초 의원의 절대 수를 계산 : <img alt="식 (1)" fo:content-width="4in" src="/files/ftp_upload/52651/52651eq1.jpg" /></li></ol> MP P 1.3) 결정rotein 농도 (브래드 포드 분석) <ol><li> 1.25 ~ 20 ㎍ / ml의에서 단백질 표준의 5 연속 희석을 준비합니다. 피펫 중복에 깨끗한 시험관에 각각의 표준 시료 용액 800 μL. 각각의 튜브에 브래드 포드 염색 시약 200 μl를 추가합니다. 믹스 그럼 5 분 동안 실온에서 품어. </li><li> 595 nm에서 흡광도를 측정한다. 표준 곡선의 선형 회귀를 이용 LMPs의 단백질 농도를 결정한다. </li></ol> 2. 기관지 조직 외식 및 LMPs 치료 참고 : 멸균 작업 환경에 특별한주의를 지불하고, 무균 실험을 다음에 사용되는 솔루션과 매체를 준비합니다. , 완전한 치유 미디어를 준비 100ml의 조직 치유 중간 (얼음 해동) 혈청과 조직 치유 중간 보충 교재의 1를 가하여 잘 혼합합니다. 기관지 조직 외식 2.1) 준비 <ol><li> 배양 전에 1cm 2의 6 영역에 흠집메스 블레이드 각각 100mm 조직 배양 접시 표면의 가장자리에 각각. 코트는 각각 배양 접시에 코팅 용액 2 ㎖와 100mm 조직 배양 접시 긁힌, 37 ° C에서 / 가습 CO 2 인큐베이터 O에서 N을 접시를 품어. 진공 잉여 솔루션을 기음과 조직 세척 매체의 15 ml로 접시를 채운다. </li><li> 동물 관리 윤리위원회의 승인을 프로토콜에 따라 공동으로 2 흡입 (5~7주 세) C57BL / 6 마우스를 안락사. </li><li> 무균는 메스, 뒤몽 초 고화질 트위터, 수술 가위로 폐 조직을 해부하다. 조심스럽게 실질 조직과 혈관을 제거합니다. 해당되는 경우, 실험실로 전송을 위해 얼음처럼 차가운 조직 세탁기 중간에 폐 조직을 놓습니다. </li><li> 또한 티슈 세척 매체에 잠긴 해부 기관지 폐 및 말초 조직으로부터 1~2.5 mm의 직경을 분리 기관지. 메스와 ~ 5mm 두께의 기관지 반지에 조각 기관지 조직. </li&gt; <li> 기관지 조각을 픽업하여 요리의 긁힌 부분에 그들을 배치 할 멸균 곡선 microdissecting의 집게로 떠 모션을 사용합니다. </li><li> 조직 세탁기 매체를 제거하고, 그들에게 요리를 준수 할 수 있도록 ~ 5 분 동안 실온에서 조각을 배양한다. </li><li> 각각의 요리에 완전 치유 중간의 10 ML을 추가 및 공기 조절 모듈 형 인큐베이터 실에 배치합니다. 높은 O 2 가스 혼합물 (70 % O 2, 25 % N 2와 5 % CO 2)와 챔버 플러시. 벤치 탑 궤도 인큐베이터에있는 챔버를 놓고 37 ℃에서 흔들어. 매체를 통해 단편 간헐적 흐르게 분당 10 사이클에서 24 시간 동안 흔들어 챔버. </li><li> 24 시간 배양 후, 도립 위상차 광학 현미경 하에서 조직 절편을 관찰한다. 이후 LMPs 치료 완료, 좋은 머리의 움직임과 활기찬 기관지 상피 세포와 기관지 외식을 선택합니다. </li></ol>2.2) LMPs 치료 <ol><li> 다음과 같이 완전한 성장 매체를 준비 : 해동 성장 매체 보충제를 얼음에 혈청 및 섬유 아세포 억제제. 혈청 성장 배지 100 ㎖에 200 μL 억제제 섬유 아세포 성장 배지 보충제 1 ml의 추가; 잘 혼합. 사용하기 전에 10 분 동안 37 ° C에서 완전 성장 배지를 따뜻하게. </li><li> 800 ㎍ / ml의 농도로 LMPs 스톡을 제조 PBS로 새로운 멸균 에펜 도르프 튜브에 절연 LMPs 희석. </li><li> 12- 웰 조직 배양 플레이트의 각 웰에 전체 성장 배지 0.5 ㎖의 추가. </li><li> 조직 배양 플레이트의 각 웰에 이전 프로토콜 (2.1)에서 곡선 microdissecting 집게로 선택한 기관지 외식을 전송합니다. </li><li> LMPs 처리 우물과 제어 우물을 식별하기 위해 적절하게 배양 플레이트 레이블. 25 μL 제어 차량 (40 ㎍ / ml의 최종 농도)도 각각 LMPs 처리에 25 μL LMPs 주식을 추가합니다 (LMP<html> 대조군 웰의 생산). </li><li> 부드러운 흔들림와 37 ° C에서 공기 조절 모듈 형 인큐베이터 실에서 배양을 계속합니다. </li><li> 24 시간 후, PBS로 절편을 3 회 세척하고 다음 단계 (4 % 파라 포름 알데히드 [PFA] 고정)를 진행합니다. </li></ol> 3. 조직 병리학 적 검사 3.1) 다음 단계로 진행하기 전에 다음 솔루션을 준비 <ol><li> 137 mM의 NaCl을, 2.7 mM의 KCl을 10 mM의 나 2 HPO 4, 1.76 mM의 KH 2 PO 4, 산도 7.4을 혼합하여 1X PBS 버퍼를 준비합니다. </li><li> 4 % PFA가 교반하면서 60 ℃에서 가열하고, 물 400 ㎖, PFA에서의 20g을 용해 제조하는 단계; 솔루션을 취소 10 M NaOH를 몇 방울을 추가합니다. 다음 1X PBS 버퍼를 추가하고 7.4에 500 ml의 부피와 산도를 조정합니다. 필터와 나누어지는; -20 ° C에서 저장합니다. </li><li> 다음 탈수 나 수분 보충 시약을 준비; 100 %, 90 %, 70 %, 50 % 에탄올, 크실렌. </li></ol>e_step &quot;&gt; 3.2) 이식편을 고정하고 조직 섹션 탈 파라핀 <ol><li> 4 % PFA 1.5 ml로 표시 microcentrifuge 관의 각 절편을 넣고 4 ℃에서 O / N을 배양한다. 1X PBS로 두 번 외식을 씻어. </li><li> 알코올 일련 이식편 탈수 (70 % 에탄올 : 각 3 회 30 분간, 90 % 에탄올 : 각 2 회 30 분 100 % 에탄올을 3 회 30 분 각 후 크실렌 : 3 회 20 분 각). 흄 후드에서 실온에서 모든 단계를 수행합니다. </li><li> 오븐에서 58 ° C에서 파​​라핀 조직 절편을 임베드. 회전식 마이크로톰을 사용하여 5 μm의 두께 조직 섹션을 준비합니다. </li><li> 56 ° C의 물을 욕조에 섹션을 플로트 한 다음 표시된 조직 학적 슬라이드에 섹션을 탑재합니다. 1 시간 동안 65 ° C에서 수동 염색 랙 및 건조에 슬라이드를 놓습니다. 슬라이드 실온에서 냉각시킵니다. </li><li> 파라핀을 제거하기 위해 10 분마다 자일 렌을 포함하는 4 연속 얼룩 요리 랙을 찍어. 셀만 후 100 %, 95 % : 크실렌을 제거하는 에탄올 시리즈 랙을 찍어다음, 80 %, 70 %,이어서 50 % 에탄올 (단계마다 5 분간) KO. 에탄올을 제거하는 5 분 동안 수돗물 랙을 씻어. </li></ol> 3.3) 헤 마톡 실린 및 에오신 (H &amp; E) 염색 <ol><li> 고정 된 조직 섹션 작업을 계속; 15 분 동안 마이어의 헤 마톡 실린 가득 염색 접시에 선반을 두십시오. 20 분 동안 헤 마톡 실린을 제거하기 위해 수돗물로 랙을 씻어. </li><li> 30 초 95 % 에탄올 30 sec.Place 증류수에 놓습니다. 1 분 동안 에오신 Y 솔루션 염색 접시에 놓습니다. 2 분마다 95 % 에탄올, 100 % 에탄올, 크실렌이 변화를 통해 탈수. </li><li> 초과 에오신가 제거되었는지 확인하기 위해 현미경으로 빠른 검사를 수행합니다. 커버 유리 커버 한 후, 각각의 슬라이드에 장착 중간 (피셔 SP15-100)의 2 ~ 3 방울을 놓습니다. </li></ol> 3.4) 현장 세포 죽음 검출 : TUNEL 분석 실험 <ol><li> 시작하기 전에, 단백질 분해 효소 K 작업 용액을 제조 : 20 ㎍ / ㎖의에10 mM 트리스 / 염산, pH가 7.4. </li><li> 3.2 절 (이식편 고정 및 조직 섹션 탈 파라핀)의 5 단계 1을 반복합니다. 탈의 H 2 O와 슬라이드를 씻어 </li><li> 10 분 동안 1X PBS로 슬라이드를 담근다. 초과 PBS를 배출합니다. 프로 테이나 제 K 작업 용액으로 RT에서 30 분 동안 조직 섹션을 인큐베이션. 린스는 1X PBS로 두 번 슬라이드. </li><li> 세포 죽음 탐지 키트의 사용 설명서에 기재된 바와 같이 TUNEL 분석을 수행한다. 설치 매체를 이용하여 장착하고 커버 글라스 수동으로 커버 슬립. </li><li> 광학 현미경 샘플을 분석 할 수 있습니다. 갈색 색상의 사멸 세포를 분석하는 프로 이미지 4.5을 ​​사용합니다. </li></ol>

<ol>
	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., 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=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, T., 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=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, S., 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=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. S., 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=Sickle+blood+contains+tissue+factor-positive+microparticles+derived+from+endothelial+cells+and+monocytes.">Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes.</a> Blood. 102 (7), 2678-2683 (2003).</li><li>Mause, S. F., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+protagonists+of+a+novel+communication+network+for+intercellular+information+exchange.">Microparticles: protagonists of a novel communication network for intercellular information exchange.</a> Circ Res. 107 (9), 1047-1057 (2010).</li><li>Yang, C., 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-proliferative+and+anti-tumour+effects+of+lymphocyte-derived+microparticles+are+neither+species-+nor+tumour-type+specific.">Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific.</a> J Extracell Vesicles. 3, (2014).</li><li>Soleti, 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=Microparticles+harboring+Sonic+Hedgehog+promote+angiogenesis+through+the+upregulation+of+adhesion+proteins+and+proangiogenic+factors.">Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors.</a> Carcinogenesis. 30 (4), 580-588 (2009).</li></ol>

저자가 공개하는 게 없다.

의원 간 크로스 토크의 활성 매개체 및 그들의 연구는 과학의 많은 분야에서 유망된다. (11)이 연구는 사멸의 T 세포주의 시험 관내 LMPs 대규모 생성을위한 상세한 프로토콜을 제시 하였다. 이러한 의원 림프구 분자의 큰 레퍼토리를 발현과 생물학적 세포 및 조직의 항상성 조절에 연루되어있다. 그러나, 서로 다른 소스에서 파생 된 LMPs 생물학적 다를 수 있습니다. 4,9,12,13 </p...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LMPs production and characterization</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CEM T cells&#160;</td>
			<td style="width:231px;">ATCC&#160;</td>
			<td style="width:124px;">CCL-119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">X-VIVO 15 medium&#160;</td>
			<td style="width:231px;">Cambrex, Walkersville</td>
			<td>04-744Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T75</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1813.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T175</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1812.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Actinomycin&#160;D&#160;</td>
			<td style="width:231px;">Sigma Chemical Co.</td>
			<td style="width:124px;">A9415-2mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PBS</td>
			<td style="width:231px;">Lifetechnologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#181;m filter</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1826.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Annexin-VCy5</td>
			<td style="width:231px;">BD Pharmagen&#160;</td>
			<td>559933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS flow solution</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">342003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fluorescent microbeads (1 um)</td>
			<td style="width:231px;">Molecular Probes&#160;</td>
			<td style="width:124px;">T8880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysterene counting beads (7 um)</td>
			<td style="width:231px;">Bangs laboratories</td>
			<td style="width:124px;">PS06N/6994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene FACS tubes</td>
			<td>Falcon</td>
			<td style="width:124px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1 ml pipet</td>
			<td style="width:231px;">Fisher</td>
			<td style="width:124px;">13-678-11B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">5 ml pipet</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:124px;">357543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">25 ml pipet</td>
			<td style="width:231px;">Ultident</td>
			<td style="width:124px;">DL-357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1,5 ml conical polypropylene micro tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">72.690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.554.205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.547.205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">50 ml high speed polypropylene copolymer tube</td>
			<td style="width:231px;">Nalgene</td>
			<td style="width:124px;">3119-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">250 ml high speed polypropylene bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">356011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay (Bradford assay)</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay standard II</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 16x100</td>
			<td>VWR</td>
			<td>47729-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 12x75</td>
			<td>Ultident</td>
			<td>170-14100005B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator&#160;</td>
			<td style="width:231px;">Mandel</td>
			<td style="width:124px;">Heracell 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Low speed centrifuge</td>
			<td style="width:231px;">IEC</td>
			<td style="width:124px;">Centra8R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed centrifuge</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Avanti J8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 250ml bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JLA16.250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 50ml tube</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JA30.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fow cytometry&#160;</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">FACS Calibur</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Spectrophotometer</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Series 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bronchial tissue explants and sections&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice (5-7 weeks old)&#160;&#160;</td>
			<td style="width:231px;">Charles River Laboratories, Inc.&#160;</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Airway PrimaCell&#8482; System:</td>
			<td style="width:231px;">CHI Scientific, Inc.</td>
			<td style="width:124px;">2-82001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Rib-Back Carbon Steel Scalpel Blades</td>
			<td style="width:231px;">Becton Dickinson AcuteCare</td>
			<td style="width:124px;">371310</td>
			<td>#10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">10003-12</td>
			<td>#7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phase-contrast inverted microscope</td>
			<td style="width:231px;">Olympus Optical CO., LTD.&#160;&#160;&#160;</td>
			<td style="width:124px;">CK2</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">high O2 gas mixture&#160;</td>
			<td>VitalAire Canada Inc.</td>
			<td style="width:124px;"></td>
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			<td height="21" style="height:21px;width:303px;">modular incubator chamber</td>
			<td style="width:231px;">Billups-Rothenberg Inc.</td>
			<td style="width:124px;">MIC-101</td>
			<td></td>
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			<td height="21" style="height:21px;">MaxQ 4000 incubated orbital shaker</td>
			<td style="width:231px;">Barnstead Lab-Line,&#160;</td>
			<td style="width:124px;">SHKA4000-7</td>
			<td></td>
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			<td height="21" style="height:21px;">12-well tissue culture plate</td>
			<td style="width:231px;">Becton Dickinson and Company</td>
			<td style="width:124px;">353043</td>
			<td></td>
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			<td height="21" style="height:21px;width:303px;">Plastic tissue culture dishes (100 mm)</td>
			<td style="width:231px;">Sarstedt, Inc.</td>
			<td style="width:124px;">83.1802</td>
			<td></td>
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			<td height="42" style="height:42px;width:303px;">Surgical scissors</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">14060-09</td>
			<td style="width:171px;">Straight, sharp, 9cm longth</td>
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			<td height="21" style="height:21px;width:303px;">Half-curved Graefe forceps</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">11052-10</td>
			<td></td>
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			<td height="21" style="height:21px;width:303px;">humidified CO2 incubator</td>
			<td style="width:231px;">Mandel Scientific Company Inc.&#160;</td>
			<td style="width:124px;">SVH-51023421</td>
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			<td style="width:231px;"></td>
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			<td height="21" style="height:21px;">&#160;Histopathological examination&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">formalin formaldehyde</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT5011</td>
			<td></td>
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			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">T555</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">ethyl alcohol</td>
			<td style="width:231px;">Merck KGaA, Darmstadt</td>
			<td style="width:124px;">EX0278-1</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;glutaraldehyde&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">G6403</td>
			<td></td>
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			<td height="21" style="height:21px;">Cacodylate</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">31533</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">microscope slides</td>
			<td style="width:231px;">VWR Scientific Inc.&#160;</td>
			<td style="width:124px;">48300-025</td>
			<td>25x75mm</td>
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			<td height="21" style="height:21px;">Xylene</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">X5-4</td>
			<td></td>
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			<td height="21" style="height:21px;">Mayer&#39;s hematoxylin</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">MHS16</td>
			<td>Funnel with filter paper&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl&#160;</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.&#160;&#160;</td>
			<td style="width:124px;">A144s-500</td>
			<td></td>
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			<td height="21" style="height:21px;">eosin&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT110116</td>
			<td>Funnel with filter paper&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permount&#8482; Mounting Medium</td>
			<td style="width:231px;">Thermo Fisher Scientific Inc.&#160;</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass coverslip</td>
			<td style="width:231px;">surgipath medical industries, Inc.</td>
			<td style="width:124px;">84503</td>
			<td>24&#215;24&#160;#1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TUNEL detection kit</td>
			<td style="width:231px;">In Situ Cell Death Detection, POD</td>
			<td style="width:124px;">11 684 817 910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">oven</td>
			<td style="width:231px;">Despatch Industries Inc.</td>
			<td style="width:124px;">LEB-1-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">rotary Microtome</td>
			<td style="width:231px;">Leica Microsystems Inc.</td>
			<td style="width:124px;">RM2145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">filter paper</td>
			<td style="width:231px;">Whatman International Ltd.</td>
			<td style="width:124px;">1003150</td>
			<td>#3</td>
		</tr>
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			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:231px;">Nikon Imaging Japan Inc.</td>
			<td style="width:124px;">E800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">staining dish complete</td>
			<td style="width:231px;">Wheaton Industries, Inc.</td>
			<td>900200</td>
			<td>including dish, rack, cover</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1.5 ml eppendorf tube</td>
			<td style="width:231px;">Sarstedt Inc.&#160;</td>
			<td style="width:124px;">72.69</td>
			<td>39x10mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital and Reciprocating Water Bath</td>
			<td style="width:231px;">ExpotechUSA</td>
			<td style="width:124px;">ORS200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffered saline&#160;&#160;</td>
			<td style="width:231px;">GIBCO</td>
			<td style="width:124px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fume hood</td>
			<td>Nicram RD Service</td>
			<td>3707E</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

세포 간 통신 세포막 유래 소체의 생물학적 역할에 대한 관심이 최근 증가하고있다. 미세 입자 (의원) 1 ㎛, 0.1 ㎛의 범위에서 직경이, 소체의 하나의 이러한 유형이며, 통상적으로 활성화 또는 사멸을 겪는 진핵 세포의 세포막에서 나타냈다. 여기에서 우리는 D. LMPs는 다단계 차동 원심 분리 과정을 통해 고립되고 유동 세포 계측법하여 특성화 티노 마이신 자극 세포 사멸 CEM T 세포에서 T 림프구에서 유래 미세 입자 (LMPs)의 생성을 설명합니다. 이 프로토콜은 또한 마우스 호흡기 차 기관지 조직 이식편 유래의 기관지 상피 세포에서의 아폽토시스 LMPs 효과를 입증하기위한 인 시츄 세포 사멸 검출 방법을 제안한다. 여기에 설명 된 방법은 체외에서 세포 사멸 림프구에서 LMPs의 풍부한 수량을 분리하기위한 재현 과정을 제공합니다. LMPs 유도이와 같이 각종 질병 모델의 특성을 평가하는 데 사용되며, 약리학 및 독성학 테스트 할 수있다. 기도 상피는 외부 환경과 하부 조직 사이의 보호 물리적 및 기능적 장벽을 제공 감안할 기관지 조직 이식편보다는 불멸화 상피 세포주를 사용기도 관 조직을 요구 수사 효과적인 모델을 제공한다.

LMPs 형광 활성화 셀 (FACS) 분석을 정렬 및 의원 (≤1 μm의)의 97 %가 넥신-V-Cy5에 긍정적 된 1 μm의 구슬 사용 정문에 의해 넥신 V 염색 (10) 특성화되었다 (그림 1A 및 1B). 일반적 LMPs 약 2.5 mg을이 프로토콜 다음 얻었다. C57BL 기관지 조직 이식편은 / 6 마​​우스는 비히클 및 LMPs 처리를 실시 하였다. 기관지 섹션의 조직 병리학 적 분석은 기관지 상피 세포의 구조적 무결성에 LMPs의 효...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

세포막 창 고 미립자 (의원) 절연 및 병태 생리 학적 효과는 다양한 모델에서 조사 할 수있는 활성 생물학적 소포입니다. 여기에서 우리는 T 림프구 (LMPs)에서 파생 된 의원을 생성하고기도 상피 세포에 자신의 아폽토시스 효과를 입증하는 방법을 설명합니다.

기도 상피 세포에 림프 미세 입자 및 아폽토시스 효과의 검출 세대

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Research

JoVE Journal

Immunology and Infection

8.9K 조회수.  University of Montréal.  세포막 창 고 미립자 (의원) 절연 및 병태 생리 학적 효과는 다양한 모델에서 조사 할 수있는 활성 생물학적 소포입니다. 여기에서 우리는 T 림프구 (LMPs)에서 파생 된 의원을 생성하고기도 상피 세포에 자신의 아폽토시스 효과를 입증하는 방법을 설명합니다. 

비디오: Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

공동 문화 모델 녹농균 Biofilms는 라이브 인간 에어웨이 세포에 들어라

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

연구

면역학 및 감염병학

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

포유류의 상피 세포에 박테리아의 접착 시험 관내 분석

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

마우스의 응용은 M 세포에 의해 박테리아 이해를 평가하는 Peyer의 패치 장 루프 분석 출혈도 잡았

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

에 보이는 빛과 젠타 마이신의 상승 효과<em&gt; Pseudomona 녹농균</em&gt; 미생물

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지<em&gt; 체외에서</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

상부 기도 면역 중재자 수준 Vivo에서 의 정량화에 대 한 점 막 안 대기의 비 침범 성 샘플링

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...