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><tbody><tr><td>LMPs production and characterization</td><td></td><td></td><td></td></tr><tr><td>CEM T cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CCL-119</td><td></td></tr><tr><td>X-VIVO 15 medium&amp;nbsp;</td><td>Cambrex, Walkersville</td><td>04-744Q</td><td></td></tr><tr><td>Flask T75</td><td>Sarstedt</td><td>83.1813.502</td><td></td></tr><tr><td>Flask T175</td><td>Sarstedt</td><td>83.1812.502</td><td></td></tr><tr><td>Actinomycin&amp;nbsp;D&amp;nbsp;</td><td>Sigma Chemical Co.</td><td>A9415-2mg</td><td></td></tr><tr><td>PBS</td><td>Lifetechnologies</td><td>14190-144</td><td></td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>Annexin-VCy5</td><td>BD Pharmagen&amp;nbsp;</td><td>559933</td><td></td></tr><tr><td>FACS flow solution</td><td>BD Bio-sciences</td><td>342003</td><td></td></tr><tr><td>Fluorescent microbeads (1 &amp;micro;m)</td><td>Molecular Probes&amp;nbsp;</td><td>T8880</td><td></td></tr><tr><td>Polysterene counting beads (7 &amp;micro;m)</td><td>Bangs laboratories</td><td>PS06N/6994</td><td></td></tr><tr><td>Polypropylene FACS tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>1 ml pipet</td><td>Fisher</td><td>13-678-11B</td><td></td></tr><tr><td>5 ml pipet</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>25 ml pipet</td><td>Ultident</td><td>DL-357551</td><td></td></tr><tr><td>1.5 ml conical polypropylene micro tube</td><td>Sarstedt</td><td>72.690</td><td></td></tr><tr><td>15 ml conical polypropylene tube</td><td>Sarstedt</td><td>62.554.205</td><td></td></tr><tr><td>50 ml conical polypropylene tube</td><td>Sarstedt</td><td>62.547.205</td><td></td></tr><tr><td>50 ml high speed polypropylene copolymer tube</td><td>Nalgene</td><td>3119-0050</td><td></td></tr><tr><td>250 ml high speed polypropylene bottle</td><td>Beckman</td><td>356011</td><td></td></tr><tr><td>Protein assay (Bradford assay)</td><td>Bio-Rad Laboratories</td><td>500-0006</td><td></td></tr><tr><td>Protein assay standard II</td><td>Bio-Rad Laboratories</td><td>500-0007</td><td></td></tr><tr><td>Test tube 16 x 100</td><td>VWR</td><td>47729-576</td><td></td></tr><tr><td>Test tube 12 x 75</td><td>Ultident</td><td>170-14100005B</td><td></td></tr><tr><td>Cell incubator&amp;nbsp;</td><td>Mandel</td><td>Heracell 150</td><td></td></tr><tr><td>Low speed centrifuge</td><td>IEC</td><td>Centra8R</td><td></td></tr><tr><td>High speed centrifuge</td><td>Beckman</td><td>Avanti J8</td><td></td></tr><tr><td>High speed rotor for 250ml bottle</td><td>Beckman</td><td>JLA16.250</td><td></td></tr><tr><td>High speed rotor for 50ml tube</td><td>Beckman</td><td>JA30.50</td><td></td></tr><tr><td>Fow cytometry&amp;nbsp;</td><td>BD Bio-sciences</td><td>FACS Calibur</td><td></td></tr><tr><td>Spectrophotometer</td><td>Beckman</td><td>Series 600</td><td></td></tr><tr><td>Bronchial tissue explants and sections&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6 mice (5-7 weeks old)&amp;nbsp;&amp;nbsp;</td><td>Charles River Laboratories, Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Mouse Airway PrimaCell&amp;trade; System:</td><td>CHI Scientific, Inc.</td><td>2-82001</td><td></td></tr><tr><td>&amp;nbsp;Rib-Back Carbon Steel Scalpel Blades</td><td>Becton Dickinson AcuteCare</td><td>371310</td><td>#10</td></tr><tr><td>Scalpel Handle</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>10003-12</td><td>#7</td></tr><tr><td>phase-contrast inverted microscope</td><td>Olympus Optical CO., LTD.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>CK2</td><td></td></tr><tr><td>high O&lt;sub&gt;2&lt;/sub&gt; gas mixture&amp;nbsp;</td><td>VitalAire Canada Inc.</td><td></td><td></td></tr><tr><td>modular incubator chamber</td><td>Billups-Rothenberg Inc.</td><td>MIC-101</td><td></td></tr><tr><td>MaxQ 4000 incubated orbital shaker</td><td>Barnstead Lab-Line,&amp;nbsp;</td><td>SHKA4000-7</td><td></td></tr><tr><td>12-well tissue culture plate</td><td>Becton Dickinson and Company</td><td>353043</td><td></td></tr><tr><td>Plastic tissue culture dishes (100 mm)</td><td>Sarstedt, Inc.</td><td>83.1802</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>14060-09</td><td>Straight, sharp, 9cm longth</td></tr><tr><td>Half-curved Graefe forceps</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>11052-10</td><td></td></tr><tr><td>humidified CO2 incubator</td><td>Mandel Scientific Company Inc.&amp;nbsp;</td><td>SVH-51023421</td><td></td></tr><tr><td>&amp;nbsp;Histopathological examination&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>formalin formaldehyde</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>HT5011</td><td></td></tr><tr><td>paraffin</td><td>Fisher scientific&amp;nbsp; International, Inc.</td><td>T555</td><td></td></tr><tr><td>ethyl alcohol</td><td>Merck KGaA, Darmstadt</td><td>EX0278-1</td><td></td></tr><tr><td>&amp;nbsp;glutaraldehyde&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>G6403</td><td></td></tr><tr><td>Cacodylate</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>31533</td><td></td></tr><tr><td>microscope slides</td><td>VWR Scientific Inc.&amp;nbsp;</td><td>48300-025</td><td>25x75 mm</td></tr><tr><td>Xylene</td><td>Fisher scientific&amp;nbsp; International, Inc.</td><td>X5-4</td><td></td></tr><tr><td>Mayer&#039;s hematoxylin</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>MHS16</td><td>Funnel with filter paper&amp;nbsp;&amp;nbsp;</td></tr><tr><td>HCl&amp;nbsp;</td><td>Fisher scientific&amp;nbsp; International, Inc.&amp;nbsp;&amp;nbsp;</td><td>A144s-500</td><td></td></tr><tr><td>eosin&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>HT110116</td><td>Funnel with filter paper&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Permount&amp;trade; Mounting Medium</td><td>Thermo Fisher Scientific Inc.&amp;nbsp;</td><td>SP15-100</td><td></td></tr><tr><td>glass coverslip</td><td>surgipath medical industries, Inc.</td><td>84503</td><td>24&amp;times;24&amp;nbsp;#1&amp;nbsp;</td></tr><tr><td>TUNEL detection kit</td><td>In Situ Cell Death Detection, POD</td><td>11 684 817 910</td><td></td></tr><tr><td>oven</td><td>Despatch Industries Inc.</td><td>LEB-1-20</td><td></td></tr><tr><td>rotary Microtome</td><td>Leica Microsystems Inc.</td><td>RM2145</td><td></td></tr><tr><td>filter paper</td><td>Whatman International Ltd.</td><td>1003150</td><td>#3</td></tr><tr><td>Microscope</td><td>Nikon Imaging Japan Inc.</td><td>E800</td><td></td></tr><tr><td>staining dish complete</td><td>Wheaton Industries, Inc.</td><td>900200</td><td>including dish, rack, cover</td></tr><tr><td>1.5 ml eppendorf tube</td><td>Sarstedt Inc.&amp;nbsp;</td><td>72.69</td><td>39x10 mm</td></tr><tr><td>Orbital and Reciprocating Water Bath</td><td>ExpotechUSA</td><td>ORS200</td><td></td></tr><tr><td>phosphate buffered saline&amp;nbsp;&amp;nbsp;</td><td>GIBCO</td><td>14190-144</td><td></td></tr><tr><td>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

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

연구

면역학 및 감염병학

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

인간 유도 만능 줄기 세포 유래 유형 2 폐포 상피 세포의 3D 구체 및 2D 공기-액체 계면 배양 생성

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

발생학

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

인간 다능성 줄기 세포로부터의 기도 상피 세포 공기-액체 계면 배양물의 생성

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

정밀 절단 라이브 폐 수지상 세포를 시각화하기 위해 마우스 폐 슬라이스

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

오존 노출 에 따른 폐포 대식세포 비세포증의 생체내 평가

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

생물학

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

바이오뱅킹을 위해 절제된 폐 조직에서 기관지 상피 세포를 분리하고 잘 분화된 공기-액체 계면 배양을 확립합니다.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

최적의 Lentivirus 생산 및 세포 배양 조건에 필요한 성공적으로 형질 도입 기본 인간 기관지 상피 세포

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

세포 내 이웃 셀을받은 사멸 세포 죽음과의 상호 작용에 의해 실행 가능한 세포에서 유도 된 이벤트를 시그널링의 식별

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...