이 작품은 바이오 메디컬 연구 및 NIH RO1 AI083426 - 01 (FW)에 우수 스미스 가족의 수상에 의해 투자되었다. 패트릭 Maschmeyer와 멜라니 Flach는 Boehringer Ingelheim 재단에서 박사 장학금 지원합니다.

C57BL / 6 생쥐 ~ 20 세 주 이상에서 사용해야합니다. 25-30g 무게 남성 마우스의 사용을 권장합니다. 별 모양의 세포의 수율은 셀 절연을 별 모양 이전에 2 개월 쥐에게 비타민 A - 농축 다이어트 먹이를 증가 수 있습니다. Balb / C 마우스에서 별 모양의 세포의 수율이 상당히 높은 반면 약 2x10 5 간장 별 모양의 세포는 하나의 C57BL / 6 마우스의 간장에서 정화 수 있습니다. 다음 프로토콜은 5 마우스로 조정됩니다. 동물 관리 및 실험이 승인 기관 애니멀 케어 및 사용위원회 프로토콜에 따라 수행되었다. 1. 마우스 간은 현장의 재관류에서 소화 효소와 함께 <ol><li> 37 ° C의 물을 욕조에 SC1 버퍼와 효소 재관류 솔루션을 따뜻하게. </li><li> 연동 펌프의 실리콘 튜브로 설정 날개 주입을 탑재합니다. </li><li> 간의 재관류 현장에서 사용되는 유량입니다 6.5ml/min의 층류 흐름을 얻기 위해 PBS로 펌프를 보정합니다. SC1 솔루션과 실리콘 튜브를 평형. </li><li> 케타민을 (90 MG / kg)과 Xylazine (10 MG / kg) 솔루션과 깊이 마취제를 투여함을 보장하기 위해 반사 손실 테스트 intraperitoneal 주사로 마우스를 마취. </li><li> 적당한 기지에 위로 향한 위치에 마우스를 수정. </li><li> 복부 피부에 세로 절개를 수행하고 복막을 폭로하기 위해 가위와 집게를 사용하십시오. </li><li> 조심스럽게 복막을 열고 포털 정맥을 노출하기 위해 동물의 왼쪽으로 복강의 내장을 이동합니다. </li><li> 포털 정맥에 주입 세트의 정맥을 넣습니다. 이 단계에 대한, stereomicroscope의 사용을 권장합니다. </li><li> 30ml SC1 솔루션으로 간의 재관류를 시작합니다. 즉시 흐름을 시작 후 베나 카바 하부를 엽니다. 간장의 성공 플러싱은 간 조직의 색깔의 손실로 표시됩니다. </li><li> 30ml Pronase 전자 솔루션으로 간을 Perfuse. 성공적으로 소화시 간 엽 (叶)이 팽창되며, lobules은 캡슐을 통해 뚜렷한 나타납니다. </li><li> 30ml Collagenase의 P 솔루션으로 간을 Perfuse. 이 시점에서 간은 그 모양을 잃고 이완증의 및 비정질 보인다 있습니다. </li><li> 신중하게 횡경막과 주변 장기에서 간장을 분리하고 그것은 얼음 70ml SC2 용액에 보관합니다. </li><li> 추가 생쥐에 대한 절차를 반복합니다. </li></ol> 2. 마우스 간은의 체외의 소화에 모든 추가 작업 단계 층류 후드에서 무균 조건 하에서 수행되어야합니다. <ol><li> 날카로운 가위를 사용하여 약 2x2x2mm 3 조각으로 간을 잘라. </li><li> Pronase E - Collagenase P 솔루션 50ml로 간 조각을 포함 70ml SC2의 정지를 결합하고 DNase I 솔루션의 1ml를 추가합니다. </li><li> 37 20 분 대한 간은을 소화 ° C가 감동하는 동안. </li></ol> 3. 밀도 기울기 원심 분리 장치 <ol><li> 6 50ml 팰컨 튜브로 70μm 셀 strainers 통해 세포 현탁액을 필터링하고 SC2 버퍼 50ml까지 추가할 수 있습니다. 600g 4 ° C.에 10 분에 대한 원심 분리기 </li><li> 조심스럽게 뜨는의 40ml를 대기음, 다음 각 튜브에 150μl DNase I 솔루션을 추가하고 세포를 resuspend. </li><li> 4 50ml 팰컨 튜브에 수영장 셀 suspensions과 600g 4시 10 분에 대한 GBSS - B, 원심 분리기로 씻어 ° C. </li><li> 조심스럽게 펠렛 각 튜브에 추가 150μl DNase I 솔루션을 방해하지 않고 최대한 뜨는의를 대기음. 튜브 당 10ml GBSS - B의 세포 알약을 Resuspend. </li><li> 수영장 2 50ml 팰컨 튜브에 세포 튜브 당 36ml의 총 볼륨 GBSS - B를 추가합니다. 각각의 튜브에 14ml Nycodenz 솔루션을 추가하고 잘 섞는다. </li><li> 총 10 튜브의 결과로 하나 12ml 기울기 원심 분리 튜브에 세포 현탁액의 10ml를 전송합니다. 부드럽게 1.5ml GBSS - B 당 튜브와 세포 현탁액을 오버레이. </li><li> 1,500그램 4에서 15을 위해 그라디언트를 원심 ° C 브레이크없이. 별 모양의 세포가 흰색 고리로서 계면에서 발견되는 반면, 그 후, hepatocytes는 튜브의 하단에 pelleted 것입니다. </li><li> 조심스럽게 별 모양의 세포를 포함하는 계면을 수확하고 GBSS - B와 함께 그들을 씻고, 600g, 4에서 원심 분리기를위한 10 분 ° C. </li><li> 뜨는을 대기음와 10 %의 열 inactivated FBS, 1 % 페니실린 / 스트렙토 마이신, 2mM L - 글루타민, 1mM 나트륨 pyruvate 및 10mM HEPES와 보충 20ml DMEM에있는 세포를 resuspend. 2x10 4 셀 / cm 2 농도와 조직 문화 flasks에 세포를 전송합니다. 37 ° C와 5% CO 2에있는 세포를 품어. </li><li> 즉시 간장 별 모양의 세포가 죽은 세포와 세포 부스러기를 씻어 (약 2 시간 준비 후) 자기편 그대로 미디어를 변경합니다. </li><li> 다음 날, 별 모양의 세포에S는 perinuclear 비타민 A - 저장 지질 vesicles과의 특성 별 모양의 형태를 개발해야합니다. </li><li> 후속 실험, 간장 별 모양의 세포는 쉽게 같은 Accutase, 예를 들어 같은 가벼운 효소 솔루션을 사용하지 않는 코팅 플라스틱 표면에서 분리 수 있습니다 </li></ol> 4. 대표 결과 : 제공된 프로토콜을 사용하여 간장 별 모양의 세포의 준비 다음, 고립된 인구의 순도는 이러한 glial fibrillary 산성 단백질의 스타와 같은 모양, perinuclear 지질 방울, 그리고 표현 (GFAP로 세포 유형의 세 가지 주요 특성을 고려하여 테스트를 수 ). 특성 세포 분리 후 간장 별 모양의 세포의 2 시간의 외관뿐만 아니라 체외 문화의 날 1 3에 대한 대표적인 사진은 그림 1에 묘사된 있습니다. 그림 2는 세포 격리 다음 3 일간 교양되었습니다 간장 별 모양의 세포에서 GFAP에 대한 대표 immunofluorescence 염색법을 보여줍니다. 간장 별 모양의 세포는 체외 문화 중에 myofibroblastic 세포로 구별. 이러한 활성 별 모양의 세포의 특성 특징은 알파 평활근 굴지 (αSMA)의 표현이다. 그림 3은 체외 문화의 날 7 활성화 별 모양의 세포에 aSMA과 마이 오신 IIA에 대한 immunofluorescence 염색법을 보여줍니다. <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="그림 1" /> 그림 1. 간성 별 모양의 세포의 특성 형태. 별 모양의 세포가 제공하는 프로토콜을 사용하여 마우스 간은으로부터 격리되었다. 이미지가 별 모양의 세포가 세포 격리 후 2 시간 (A, B)뿐만 아니라 1 일 (C)와 체외 문화의 3 일 (D)에 대한 묘사. 간장 별 모양의 세포가 perinuclear 사이트에서 지질 vesicles 높은 금액을 전시하고 체외 문화의 첫 번째 일 (확대 200x) 동안 자신의 특유의 아스트랄과 같은 형태를 취득. <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="그림 2" /> 그림 2. 간성 별 모양의 세포는 특히 간장에 GFAP을 표현. 별 모양의 세포는 체외 문화의 날 3 격리 및 immunofluorescently GFAP (적색)에 얼룩했다. 세포 핵은 파란색 (Hoechst는 얼룩)에 그려져있다. <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="그림 3" /> 그림 3. 간 별 모양의 세포가 myofibroblasts로 구분. C57BL / 6 마​​우스에서 분리된 간장 별 모양의 세포는 7 일 교양되었습니다. 그 후, 그들은 챔버 슬라이드로 전송하고 aSMA (빨간색으로 표시)과 마이 오신 IIA (녹색에 묘사된)에 얼룩했다. 세포 핵은 Hoechst (파란색)과 counterstained되었습니다. <table border="1"><tbody><tr><td colspan="2"> SC1 버퍼 </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td> 포도당 </td><td> 900mg </td></tr><tr><td> HEPES </td><td> 1M 재고 솔루션 10 ML </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> 나 2 HPO 4 X 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> 아니 2 PO 4 X H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> 페놀 레드 </td><td> 6mg </td></tr><tr><td> DH 2 O </td><td> 1리터까지 작성 </td></tr><tr><td colspan="2"> SC2 버퍼 </td></tr><tr><td> CaCl 2 X 2 시간 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 1M 재고 솔루션 10ml </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> 나 2 HPO 4 X 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> 아니 2 PO 4 X H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> 페놀 레드 </td><td> 6mg </td></tr><tr><td> DH 2 O </td><td> 1리터까지 작성 </td></tr><tr><td colspan="2"> GBSS - 버퍼 </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> CaCl 2 X 2 시간 2 O </td><td> 225mg </td></tr><tr><td> 포도당 </td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 X 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 X 7 H 2 O </td><td> 70mg </td></tr><tr><td> 나 2 HPO 4 X 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> 페놀 레드 </td><td> 6mg </td></tr><tr><td> DH 2 O </td><td> 1리터까지 작성 </td></tr><tr><td colspan="2">GBSS - B 버퍼 </td></tr><tr><td> CaCl 2 X 2 시간 2 O </td><td> 225mg </td></tr><tr><td> 포도당 </td><td> 991mg </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 X 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 X 7 H 2 O </td><td> 70mg </td></tr><tr><td> 나 2 HPO 4 X 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> 페놀 레드 </td><td> 6mg </td></tr><tr><td> DH 2 O </td><td> 1리터까지 작성 </td></tr><tr><td colspan="2"> Pronase 전자 재관류 솔루션 </td></tr><tr><td> Pronase E </td><td> 100mg (4000 PU / MG 분) </td></tr><tr><td> SC2 버퍼 </td><td> 200ml </td></tr><tr><td colspan="2"> Collagenase P 재관류 솔루션 </td></tr><tr><td> Collagenase P </td><td> 85mg (1.78 U / MG lyo) </td></tr><tr><td> SC2 버퍼 </td><td> 200ml </td></tr><tr><td colspan="2"> Pronase E - Collagenase P 솔루션 </td></tr><tr><td> Pronase E </td><td> 50mg (4000 PU / MG 분) </td></tr><tr><td> Collagenase P </td><td> 85mg (1.78 U / MG lyo) </td></tr><tr><td> SC2 버퍼 </td><td> 50ml </td></tr><tr><td colspan="2"> DNase I 솔루션 </td></tr><tr><td> DNase I </td><td> 6mg (ca. 2000U/mg) </td></tr><tr><td> GBSS - B </td><td> 3ml </td></tr><tr><td colspan="2"> Nycodenz 솔루션 </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS - A </td><td> 28ml </td></tr></tbody></table> 표 1. 버퍼와 간장 별 모양의 세포의 분리에 필요한 효소 솔루션을 제공합니다. 모든 버퍼의 산도는 7.3-7.4로 조정해야합니다. 또한, 모든 버퍼의 무균 여과 권장합니다. 해당 효소 활동에 따라 사용되는 효소의 양을 적응하는 것이 중요합니다.

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관심 없음 충돌 선언하지 않습니다.

간장 별 모양의 세포는 간장에 필수적인 생리와 pathophysiological 프로세스를 조절. 또한, 별 모양의 세포가 그들에게 간장 면역 반응의 중요한 구성 요소를 렌더링, 항원 제시 속성을 보유하고 있습니다. 간장 별 모양의 세포가 간 세포에있는 전체 번호 10~15%을 구성하지만, 이러한 세포의 격리는 쭉 내려 가래의 perisinusoidal 공간에서 자신의 로컬 라이제이션으로 인해 도전이다. ...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 70μm 셀 스트레이너 </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCL 2 X 2 시간 2 O </td><td> 시그마 - 알드리치 </td><td> C3306 </td></tr><tr><td> Collagenase P </td><td> 로체 </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11,960 </td></tr><tr><td> DNase I </td><td> 로체 </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21-031 - CV </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> 태아 소 혈청 </td><td> Gibco </td><td> 16,000 </td></tr><tr><td> 포도당 </td><td> 시그마 - 알드리치 </td><td> G7528 </td></tr><tr><td> 기울기 원심 분리 튜브 </td><td> 그라 이너 바이오 원 </td><td> 163160 </td></tr><tr><td> HEPES </td><td> Gibco </td><td> 15,630 </td></tr><tr><td> KCL </td><td> 시그마 - 알드리치 </td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td> 시그마 - 알드리치 </td><td> P9791 </td></tr><tr><td> L - 글루타민 </td><td> Gibco </td><td> 25,030 </td></tr><tr><td> MgCl 2 6H 2 O X </td><td> Fluka </td><td> 63,068 </td></tr><tr><td> MgSO 4 7H 2 O X </td><td> 시그마 - 알드리치 </td><td> M2773 </td></tr><tr><td> 나 2 HPO 4 X 2 시간 2 O </td><td> Fluka </td><td> 71,643 </td></tr><tr><td> NaCl </td><td> 시그마 - 알드리치 </td><td> S3014 </td></tr><tr><td> 아니 2 PO 4 X H 2 O </td><td> Fluka </td><td> 71,507 </td></tr><tr><td> NaHCO 3 </td><td> Fluka </td><td> 71,628 </td></tr><tr><td> Nycodenz </td><td> Accudenz AG </td><td> AN7050/BLK </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Gibco </td><td> 15,140 </td></tr><tr><td> 연동 펌프 </td><td> 콜 파머 </td><td> HV - 7523-70 </td></tr><tr><td> 페놀 레드 </td><td> 시그마 - 알드리치 </td><td> P4633 </td></tr><tr><td> Pronase E </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> 실리콘 튜브 </td><td> Masterflex </td><td> HV - 96440-14 </td></tr><tr><td> 나트륨 Pyruvate </td><td> Gibco </td><td> 11,360 </td></tr><tr><td> 조직 문화 플라스크 (25cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> 빙게드 주입 세트 </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

간장 별 모양의 세포 스타 같은 형태의 간 세포 거주하고 간 사인 내피 세포와 hepatocytes 1,2 사이에 쭉 내려 가래의 공간에 위치하고 있습니다. 별 모양의 세포는 골수 엽 성의 전구 물질에서 파생 및 토탈 바디 비타민 A 1, 2의 80 %를 저장할 수 있습니다. 활성화되면, 별 모양의 세포 따라서 간 섬유증 3 기여, 세포외 기질을 생산 myofibroblasts로 구분. 계약을 자신의 능력에 따라 myofibroblastic 별 모양의 세포가 포털 고혈압 4 연관된 혈관 음색을 조절할 수 있습니다. 최근, 우리는 간장 별 모양의 세포가 강력한 항원 제시 세포이며, NKT 세포뿐만 아니라, 기존의 T lymphocytes 5 활성화할 수 있습니다 보여주었다. 여기 마우스 간부터 간장 별 모양의 세포의 효율적인 준비 방법을 제시한다. 그들 perisinusoidal 현지화으로 인해, 간장 별 모양의 세포의 격리는 여러 단계로 이루어집니다. 쭉 내려 가래의 공간에서 고립에 액세스할 별 모양의 세포를 렌더링하기 위해서는 마우스 간은이 소화 효소 Pronase E와 Collagenase P. 다음과 재관류로 현장에 perfused 있으며, 간 조직은에 Pronase E와 Collagenase P와 함께 추가적인 효소 치료를 받게됩니다 체외. 이후, 방법은 간장 별 모양의 세포에 비타민 A - 저장 지질 방울의 엄청난 금액을 활용합니다. 이 기능은 8퍼센트 Nycodenz 기울기에 원심 분리하여 다른 간장 세포 유형에서 별 모양의 세포의 분리 수 있습니다. 프로토콜 여기에서 설명한은 별 모양의 세포의 매우 순수하고 동질적인 인구 산출. 준비 순도는 이전 형광 현미경이나 유동세포계측법에 의해 분석 마커 분자 glial fibrillary 산성 단백질 (GFAP)에 대한 염색법에 의해 평가하실 수 있습니다. 또한, 가벼운 현미경은 지질 방울의 높은 금액을 가지고 있고, 별 모양의 간장 별 모양의 세포의 독특한 모양을 보여준다. 함께 찍은, 우리는 그들의 대표적인 형태학의 외관 이미지와 별 모양의 세포 실체를 정의하는 데 도움이 GFAP 표현을 포함한 간장 별 모양의 세포의 효율적인 격리에 대한 자세한 프로토콜을 제시한다.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

여기 마우스 간부터 간장 별 모양의 세포의 격리를 위해 방법을 설명합니다. 별 모양의 세포 정화 들어, 마우스 간은이 소화되게하여<em&gt; 현장에서</em&gt;과<em&gt; 체외에서</em&gt; 밀도 기울기 원심 분리하기 전에 pronase - collagenase 처리에 의해. 이 기술은 고도로 순수한 간장 별 모양의 세포를 산출.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Research

JoVE Journal

Immunology and Infection

33.5K 조회수.  Harvard Medical School. 여기 마우스 간부터 간장 별 모양의 세포의 격리를 위해 방법을 설명합니다. 별 모양의 세포 정화 들어, 마우스 간은이 소화되게하여 현장에서과 체외에서 밀도 기울기 원심 분리하기 전에 pronase - collagenase 처리에 의해. 이 기술은 고도로 순수한 간장 별 모양의 세포를 산출.

비디오: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

전체 리프 이미징을 사용하여 박테리아 세포를 라이브 Stomatal 응답을 평가

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

연구

면역학 및 감염병학

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Waterborne를 검색하는 접선 흐름 빈 섬유 Ultrafiltration 및 열 분해 단계를 사용 수정일 EPA 방법 1,623<em&gt; Cryptosporidium</em&gt;과<em&gt; Giardia SPP.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

나이브 CD4의 아데노 바이러스 형질은 Treg의 분화를 연구하는 세포를 T

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

항원 중심의 성 대장염 모델의 개발은 항원이 T 세포에 세포를 제시하여 항원의 프리젠 테이션을 공부하기

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

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 ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

혈액 방울로부터의 신속한 호중구 화학 주 화성 분석을위한 올 온칩 (all-on-chip) 방법

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

개발 및 검증 磁 단일 분자의 배열 디지털 효소 연결 된 Immunosorbent 분석 결과 인간 인터페론-α에 대 한

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...