저자는 기술 지원을 SLO 플라스미드와 조나단 프랭크의 관대 한 선물 anthrolysin O, 마이클 Caparon의 관대 한 선물 리처드 휴식을 감사드립니다. 이 작품은 NIH 보조금 T32CA82084 (박), 그리고 R01AI072083 (RDS)에 의해 재정 지원되었다.

1. Streptolysin O의 정화 (SLO) <ol><li> 0.5 L의 LB의 국물에 플라스미드 pBADgIII-SLOhis 9 포함 BL21 GOLD 세포의 20 ML 밤 문화의 예방 500 μl 50 MG / ML 암피실린을 추가합니다. 37 ° C에서 225 rpm으로 문화를 흔들어 OD 600까지 = 0.6은, 보통 1.5 시간을 ~. 10,000 5 분 XG에서 1 ML 박테리아를 (T 0으로 간주) 원심 분리기, 140 μl 1X SDS 샘플 버퍼 / 1 OD에서 펠렛을 분해하고 단백질 순도 분석에 전단 DNA에 sonicate. </li><li> 문화 5 ML 20 % arabinose의 추가와 박테리아를 유도, 3 시간에 대한 온도는 실온에서 225 rpm으로 흔들. 박테리아의 1 ML를 수집 원심 분리기 등 위의 순도 분석을위한 T 3 번 지점에 대한 1X SDS 샘플 버퍼에 용해. </li><li> 500 ML의 원심 분리기 병에 남아있는 세균을 수집, 4 ° C (8,500 RPM Sorvall GS-3 회)에서 12 분에 12,000 XG를 돌리다. -80에서 표면에 뜨는, 저장 펠릿을 가만히 따르다 필요한 경우 더 이상 ° C 하룻밤 나. </li><li> FR을 Resuspend10 ML 얼음 속에서 ozen 펠릿은 Lyse / 버퍼를 씻어 (50 MM 아니 2 PO 4, 300 MM NaCl, 10 MM 이미 다졸, 산도 8.0) 400 μl 25% 튼 X-100, 100 μl 100 MG / ML 라이소자임, 50과 보충 μl 200 밀리미터 phenylmethylsulfonyl 플루오르 화 (PMSF). 50 ML 오크 릿지 관에 resuspended 펠릿을 전송합니다. </li><li> 30 초 간격으로 얼음에 30 초에 5 번 lysate Sonicate. 스핀은 4 ° C (18,000 RPM Sorvall SS-34 회)에서 20 분에 39,000 XG에서 오크 리지 관 lysate를 sonicated. </li><li> lysate가 회전하는 동안 Lyse / 4에서 5 분 동안 1,200 rpm으로 버퍼와 스핀을 씻으 ° 10 ML 1 개 ML 니켈 NTA 아가로 오스를 씻어 C. (모든 자세한 세차 / elutions이 상황에서 원심 분리를 사용하여). 세탁을 대기음. </li><li> 단계 1.5에서 니켈 NTA 아가로 오스에 박테리아 표면에 뜨는을 추가, 4에서 2.5 시간에 부드럽게 흔들 ° C. 표면에 뜨는 수집 봐. 10 μ 4X SDS 샘플 버퍼를 표면에 뜨는 30 μl을 결합하고 순도 분석에 저장합니다. 버퍼를 Lyse / 쉐이브와의 구슬 4 번 씻으십시오트리스 / 소금 버퍼 (50 MM 트리스, 300 MM NaCl, 산도 8.0)에 CE. </li><li> , 구슬에 1 ML의 용출 버퍼 (트리스 / 소금 버퍼 250 MM 이미 다졸) 5 μl 1 M dithiothreitol (DTT)을 추가 10 분 동안 얼음에 잠복기, 회전하고 표면에 뜨는를 수집하여 3 회를 Elute. SLO는 매우 산화 환원에 민감한 단백질이며, 항상 얼음에 보관해야합니다. 심지어 짧은 기간 동안, 37 ° C로 예열하면 일단 감소, 단백질이 빠른 속도로 그 활동을 잃게됩니다. </li><li> 4에 1 분에 10,000 XG에서 용출 버퍼, 스핀에 500 μl polymixin - 복합 아가로 오스을 씻으 500 μl의 ° C와 resuspend. 독소를 고갈하려면 각 용출에 200 μl 구슬을 추가하고 30 분에 대해 4 ° C를 흔들. 1 4의 분 ° C, 그리고 표면에 뜨는을 저장 10,000 XG에 스핀. SDS-PAGE 분석을위한 10 μl 4X SDS 샘플 버퍼 각 용출의 30 μl 조화를 이루고 있습니다. </li><li> SDS-PAGE에 의해 elutions의 순수성을 테스트합니다. 종기 샘플 10 % 젤에 5 분 및로드 10 μl에 95 ° C에서 젤 분석에 저장되었습니다. GE를 얼룩단백질을 시각화 할 수 Coomassie 함께하는 (그림 2A). SLO 69 kDa입니다. (일반 수율은 4 MG / 처음 두 elutions에 ML이지만, 박테리아 스트레인과 플라스미드가 사용에 따라 다를 수 있습니다) 단백질 농도를 결정하기 위해 브래드 포드 분석을 수행합니다. </li><li> 각 용출의 용혈성 활동 (아래)를 테스트합니다. 순도, 집중력 및 용혈성 활동이 만족 풀 elutions. 단일 사용 aliquots (일반적으로 5-10 μl)에 나누어지는의 SLO는 -80에서 드라이 아이스 및 매장 ° C.에 고정 </li></ol> 2. 용혈성 분석 <ol><li> RBC 분석 완충액 (0.3 % 소 혈청 알부민 (BSA), 2 MM CaCl 2, 10 밀리미터 Hepes, 산도 7.4), 5 분에 1,200 rpm으로 회전에 양 적혈구 (적혈구)을 씻어, 그리고 2.5 %의 적혈구시에 resuspend 10 ML RBC 분석 버퍼. </li><li> / 잘 얼음에 96도 V-바닥 판에 10 μl RBC 분석 버퍼를 추가합니다. 순차적으로 중복 된 각 용출을 희석. 일반적인 희석 범위는 1:1,000으로 1:512,000 있습니다. 10을 더 독소가있는 3 개의 우물과 3 우물을 포함μl 2% 튼 각각 최소 및 최대 우물과 같은 X-100,. </li><li> 37 플레이트 및 배양을 맡아 ° C 30 분. 5 분에 1,200 rpm으로 원심 분리기. 평면 바닥 96 잘 판에 표면에 뜨는의 70 μl를 전송합니다. 405 번을 읽어 보시기 바랍니다. 한 단위는 적혈구의 50 % 용해에 필요한 독소의 희석입니다. 독소 활동은 1 단위 * 희석 factor/0.01 ML입니다. </li></ol> 3. 셀 Lytic 분석 <ol><li> 수확 세포, 수, 스핀. 2x10에 Resuspend 버퍼에 6 / ML R / B (2 MM과 RPMI CaCl 2, 0.5 % BSA) 및 20 μg / ML의 propidium의 요오드화물. 96 - 웰 V-바닥 판에 100 μl 세포를 추가합니다. </li><li> 직렬 셀에 100 μl 독소 또는 100 μl 버퍼 R / B을 추가, 버퍼 R / B에 최종 농도를 2 배에 SLO를 희석. 배양 5 분 37 ° C. U / ML 2000에서 31.25 U / ML에 일반적인 최종 농도 범위. </li><li> 흐름 cytometer에 세포를 실행하고 phycoerythrin (PE)에 대한 필터 데이터를 수집합니다. 한 로그 근무 transiently permeabilized cel을 나타냅니다이게 3 로그 근무 시간이 죽은 세포에게 4 나타내며. 다음과 같이 실험 (% PI 높은 EXP)에서 컨트롤에 죽은 세포의 비율 (% PI 높은 CTL)을 차감하여 세포의 특정 용해를 계산 : 특정 용해 = (% PI 높은 EXP - % PI 높은 CTL) / (100 - % PI 높은 CTL) * 100 </li></ol> 4. 문화 요리의 세포에 독소의 인도를 Micropipette <ol><li> 콜라겐 - 코팅 유리 하단 35mm 요리에 대한 실험 판 2x10 5 대 식세포하기 전에 어느 날. </li><li> 현미경과 microinjector를 켜십시오. 온수 무대 시간이 37 ° C.에 따뜻하게 할 수 있도록 허용 현미경의 선택은 일반적으로 로컬 사용할 수 있습니다 무엇 이냐에 따라 달라집니다. 현미경은 37 요리를 가열의 단계 수, 역 단계가 필요 ° C, 선택한 염료, 그리고 물리적 microinjector을 연결하는 공간에 적합한 여기 / 방출 필터 큐브. 버트 란드 렌즈microinjection에 도움이되지만 필요하지 않습니다. 현미경을 유도하는 컴퓨터가 데이터를 수집하고 저장할 충분한 메모리가 필요합니다. </li><li> 37 번 염색과 30 분에 라벨 세포 ° C. 검정에 따라 라벨은 5 μl Fura2 1 ML 전체 미디어에서 오전 1 ML PBS 또는 2 μl calcein에서 오전 짓을 할 수 있습니다. Fura2를 들어, 여기 / 방출은 510분의 340 나노 미터와 510분의 380 nm의에 대한 수집 및 380분의 340 신호의 비율은 칼슘 유량을 결정합니다. ethidium homodimer는 620분의 525 nm의이며, APC는 660분의 650 nm의 상태에서 calcein를 들어, 여기 / 방출은 515분의 495 nm의 것입니다. 다른 레이블도 선택 될 수있다. </li><li> 한 ML RPMI에서 PBS와 장소가있는 세포를 씻어은 2 MM CaCl 2 보충. 현미경에 장착합니다. </li><li> 4 ° C.에 20,000 XG 10 분에 독소와 dextran 물에, 그리고 원심 분리기를 희석 일반적으로, 1 μl SLO과 4 μl 10 MG / ML dextran-555은 6 μl 물에 희석되어 있습니다. Dextran 또는 다른 형광 유체 상 분자는 펨토 - 끝이 막힌되어 있지 않은지 확인하는 데 사용하고, 인제합니다CTS는 원하는. </li><li> microloader를 사용하여 희석 독소 0.07 μl와 함께 후방에서 펨토 - 팁을로드합니다. </li><li> microinjector에 펨토 - 팁을로드합니다. 팁은 모든 방향으로 이동하는 방 세포의 중심을 통해 앉아 있도록 주입기의 각도를 조정합니다. Z-제한을 해제합니다. 사출 설정은 20 PSI 백 압력 120 PSI에 0.5 초에 주입해야합니다. 이 매체를 입력 할 때까지 팁을 낮 춥니 다. </li><li> 버트 란드 렌즈, 센터에게 팁을 사용하고이 가까운 셀 인하되기 때문에 팁을 따르십시오. 일단 초점을 잃어, 일반 광학으로 전환합니다. 바늘의 그림자가 필드에 분명해야합니다. 셀 위에 집중하고 초점이 될 때까지 바늘을 낮 춥니 다. 세포에 다시 초점을주의 깊게 셀에 인접한 바늘을 가져. 분사에 대한 Z-한도를 설정합니다. 원하는 위치로 팁을 이동 영상을 개시 원하는 시점에서 독소를 출시 할 주입. 바늘을 올려, 세포의 새로운 영역으로 이동 주입. 모든 원치 않는을 방지하기 위해 홈 위치로 바늘을 이동바늘의 독소 누출. </li></ol>

<ol>
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관심 없음 충돌이 선언 없습니다.

기술은 박테리아 독소에 대한 면역 세포의 반응을 시험 할 수 여기에 설명했다. 가장 중요한 단계는 독소의 처리와 주입합니다. 독소 활동도의 취약성으로 인해 같은 준비의 다른 aliquots 사이에 매우 변수가 될 수 있습니다. 이 중 참조 셀 라인 또는 적혈구에 대한 독소의 각 나누어지는을 테스트하거나 독소의 그라디언트를 사용하여 필요로. 독소 그라디언트는 등 micropipette에 의해 전달, 실시간?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 니켈 NTA 아가로 오스 </td><td> Qiagen </td><td> 30,210 </td><td></td></tr><tr><td> polymixin - 아가로 오스 </td><td> 시그마 </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba 탈염하다 스핀 컬럼 </td><td> 어부 </td><td> PI-89891 </td><td></td></tr><tr><td> 양 적혈구 </td><td> 어부 </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> 심판이 9 참조 </td></tr><tr><td> Cy5 monoreactive 염료 </td><td> GE 헬스 케어 </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> 생명 기술 </td><td> F1221 </td><td></td></tr><tr><td> Calcein AM / 장군님hidium homodimer </td><td> 생명 기술 </td><td> L3224 </td><td></td></tr><tr><td> 안티 CD11c-APC </td><td> BD Biosciences </td><td> 550261 </td><td></td></tr><tr><td> 콜라겐 - 코팅 유리 바닥 요리 </td><td> Mattek </td><td> P35GCol - 1.5-10-C </td><td></td></tr><tr><td> 펨토 - 팁 II </td><td> 어부 </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> 어부 </td><td> E5242956003 </td><td></td></tr><tr><td> dextran 알렉사 555 </td><td> 생명 기술 </td><td> D34679 </td><td></td></tr><tr><td> Injectman NI이 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> 특정 시약 및 equipm의 표 1. 목록 및 소스다닐이 필요 해요. 회사 카탈로그 번호와 함께이 프로토콜에서 사용되는 특정 장비와 시약이 나열되어 있습니다. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 버퍼 </td><td> 구성 </td><td> 사용 단계 </td></tr><tr><td> 세탁 / Lyse </td><td> 50 ㎜ 아뇨 2 PO 4 300 MM NaCl 10 MM 이미 다졸, pH를 8.0 </td><td> 1.4 </td></tr><tr><td> 트리스 / 소금 </td><td> 50 ㎜ 트리스, 산도 8.0 300 MM NaCl </td><td> 1.7 </td></tr><tr><td> 용출 버퍼 </td><td> 50 ㎜ 트리스, 산도 8.0 300 MM NaCl 250 MM 이미 다졸 </td><td> 1.8 </td></tr><tr><td> RBC 분석 버퍼 </td><td> 0.3 % BSA 이 MM CaCl 2 10 MM HEPES, pH를 7.4 </td><td> 2.1 </td></tr><tr><td> 버퍼 R / B </td><td> RPMI 세포 배양액 이 MM CaCl 2 0.5 % BSA </td><tD&gt; 3.1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> 이 프로토콜에 사용되는 버퍼의 표 2. 목록입니다. 사용되는 버퍼는 자신의 구성과 그들이 프로토콜에 사용되는에있는 첫 번째 단계가 나열됩니다. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

세균 독소는 세포 내용과 외부 환경으로부터 물질의 유입을 누설 할 수 있도록 모공을 형성 세포막에 콜레스테롤에 바인딩. 셀은 운영 중 막 복구 프로세스가 필요합니다이 모욕, 복구, 또는 다른 독소 노출과 셀 타입 1의 양에 따라 죽을 수 있습니다. 또한 이러한 독소는 프로 염증성 크린 시토 킨 2 배열을 생산 식세포 등의 면역 세포의 활성화를 통해 감염된 호스트에 강한 염증 반응을 유도. 대부분의 그람 양성 세균은 대부분 uncharacterized 메커니즘을 통해 독성에 기여하는 표시되었습니다 콜레스테롤 구속력이 독소를 생성합니다. 이러한 독소에 노출 세포의 플라즈마 막에 Morphologic 변화는 본질적인 세포 방어를 제안, 세포 공간으로 발산 할 수 있습니다 콜레스테롤 강화 표면 돌기들에 대한 자신의 격리를 포함메커니즘 3,4. 이 과정은 신진 대사 활동의 부재에있는 모든 세포에서 발생, 화학 고정 (4) 후 EM을 사용하여 시각화 할 수 있습니다. 이러한 독소 노출에 대한 응답으로 염증을 중재 대 식세포와 같은 면역 세포에서 유도 막 소포는 IL-1 가족의 크린 시토 킨를 포함하도록 제안하고 독소를 흘리는 이러한 프로 염증성 크린 시토 킨 5,6,7을 전파 모두에 책임이있을 수 있습니다. 모두 8 caspase-1에 의존 프로세스입니다으로 IL-1β 자료 및 세포 죽음의 특정 유형 사이의 링크를 칭했다 pyroptosis이 제안되었습니다. 막 소포의 독소 바인딩, 개구, 시토 킨 릴리스, 그리고 잠재적으로 세포 사망을 포함이 대 식세포 반응의 복잡성을 해결하기 위해, 우리는 다음과 같은 독소 세포의 상호 작용 실시간으로 시각화 할 수 있도록 라벨 기술과 형광 현미경 방법을 개발 장애와 죽음의 측정 (그림 1). 사용라이브 셀 이미징의 다른 기술의 한계로 인해이 필요합니다. 유동 세포 계측법은 개별 세포의 고해상도, 실시간 시각화를 제공하지 않습니다 동안 생화학 접근 방법은 개별 셀에서 발생하는 효과를 확인할 수 없습니다. 여기에 설명 된 방법은 세포에서 복잡한 phenotypic 변화를 포함하는 다른 자극에 의​​해 유도 반응의 운동 분석에 적용 할 수 있습니다.

일반적으로 10 7 -10 8 U / ML SLO은 4 MG / ML의 단백질 농도를 구할 수 있습니다. 세포 용해에 필요한 독소의 양이 세포 유형에 따라 다르며, 보통 125-500 U / ML SLO (그림 2B)입니다. 다른 사람들이 (특히 T 세포 라인) 더 민감하지만 식세포와 같은 세포 유형 (4,000 U / ML) 더 내성이 될 수 있습니다. 이러한 민감한 상업적으로 사용할 수 SLO와 일치합니다. 독소 활동은 독소의 각 배?...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

재조합에서 콜레스테롤 바인딩 독소 streptolysin O를 정화하기위한 방법<em&gt; E. 대장균</em진핵 세포를 사는 바인딩 독소&gt; 및 시각화가 설명되어 있습니다. 독소의 현지화 배달 독소 생물학의 소설 측면을 드러내는 대상 셀에 신속하고 복잡한 변화를 유도한다.

라이브 세포 형광 현미경을 사용하여 박테리아 독소 유도 응답의 시각화

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K 조회수.  University of Pittsburgh School of Medicine. 재조합에서 콜레스테롤 바인딩 독소 streptolysin O를 정화하기위한 방법 E. 대장균 및 시각화가 설명되어 있습니다. 독소의 현지화 배달 독소 생물학의 소설 측면을 드러내는 대상 셀에 신속하고 복잡한 변화를 유도한다.

비디오: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

HIV와 라이브 공촛점 현미경의 형광 클론을 사용하여 HIV의 머릿속 휴대 전화 전송

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

연구

면역학 및 감염병학

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 ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

라이브 셀 이미징 바실러스 subtilis과 연쇄상 구균 pneumoniae 자동 시간 저속 현미경을 사용하여

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

의 단일 세포 분석 바실러스 subtilis</em형광 현미경 및 유동세포계측법 사용> Biofilms

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

곰팡이 병원체 Phagocytosis의 라이브 셀 비디오 현미경

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

소에 이펙터 및 메모리 T 세포 반응을 평가하기위한 장기 배양 인터페론-γ 효소 링크 된 Immunospot 분석의 응용 프로그램

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

의 생리적 반응을 측정<em&gt; 초파리</em&gt; 라이브 칼슘 이미징을 사용하여 지질 페로몬에 감각 뉴런

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

단일 세포 형광 현미경 검사 법에 의해 Efferocytosis의 정량화

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.