이 연구는 NIH 상 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733에 의해 부분적으로 지원되었다.

모든 동물의 작업은 규정과 의학의 알버트 아인슈타인 대학 동물 연구소의 지침에 따라 이루어졌다. C.의 1 성장 조건 콕 쿠스 네오 포르 만 H99 (혈청 형 A) <ol><li> 개별 C. 성장 사부로 한천 접시에 콕 쿠스 네오 포르 만 스 (CN) 식민지. </li><li> 어느 날 이전에 실험 한 식민지를 선택하고 사부로 배지 10 ㎖를 접종. </li><li> 문화가 250 rpm의 속도로 흔들어 37 ° C에서 하룻밤 성장하도록 허용합니다. </li></ol> 2 분리 및 C57에서 차 뼈 대 식세포의 제조 / BL6 마우스 <ol><li> 더 이상 호흡까지 CO 2 챔버 내에 동물을 배치하여 마우스를 안락사. 두 번째 조치로, cervically 해부를 시작하기 전에 목을 탈구. 70 %의 에틸 알코올로 몸 전체를 소독. </li><li> 멸균 DMEM 모두 대퇴골 및 경골과 장소 뼈를 제거합니다. </li><li> 여분의 조직과 근육 드를 사용하여 제거뼈가 완전히 깨끗해질 때까지 licate가 정리합니다. </li><li> 70 % 에틸 알코올을 포함하는 페트리 접시 만 그대로 뼈를 삽입하고 관련 미생물을 죽이기 위해 3 분 동안 배양한다. 멸균 DMEM과 페트리 접시에 뼈와 장소를 제거합니다. </li><li> 조심스럽게 뼈의 끝을 잘라. 빈 50 ML 원뿔 튜브 얼음에 배치하고 각 뼈를 통해 25 G 바늘을 사용하여 차가운 DMEM 10 ㎖ 조심스럽게 세척을 통해 뼈를 잡습니다. 주 : 각 마우스가 4 뼈 (대퇴골 두, 두 경골)를 얻을 것입니다. 따라서 한 마우스 세포 현탁액의 약 40 mL로 수득한다. </li><li> 650의 X g에서 10 분 동안 실온에서 원심 분리 세포 현탁액. </li><li> 이 원심 중에 준비하고 필터 골수 대 식세포는 20 % L929, 10 % 소 태아 혈청, 10 % NCTC-109, 1 % 펜 연쇄상, 1 % HEPES, 1 % L-글루타민이 보충 된 DMEM으로 구성되어 용지를 공급하는 살균 비 필수 아미노산 1 %, 0.1 %, 2 - 메르 캅토 에탄올. </li><li> 용지를 넣기 10 mL로 재현 탁 결과 펠릿. 세포 suspens 합격세포 덩어리를 중단하고 큰 부스러기를 제거하기 위해 70 μm의 셀 스트레이너를 통해 이온. </li><li> 플레이트 (10 판의 총) 조직 배양에 사용하도록 지정된 배양 접시에서 먹이 미디어의 10 ML에 세포 현탁액 1 ㎖. </li><li> 세포가 10 % CO 2와 37 ° C에 부착 할 수 있습니다. , 일 7 일 3 일에 신선한 공급 용지 5 ㎖를 추가 완전히 공급 매체 및 대 식세포가 사용될 준비가 교체. </li><li> 이전 실험에 날, 먹이 미디어를 제거하여 플레이트에서 대 식세포를 분리하고 플레이트에 시약을 제거 부드러운 세포의 5 ㎖를 추가합니다. </li><li> 37 ° C에서 5 ~ 10 분 동안 품어 부드러운 피펫 팅으로 대 식세포를 제거합니다. </li><li> 10 분 동안 650 XG에 원심 분리하여 펠렛 세포. </li><li> 미디어를 공급하는 1 ㎖에 재현 탁 펠렛. 1:20 희석하여, 상 혈구 세포 현탁액 10 ㎕를 피펫 팅에 의해 대 식세포의 농도를 계산한다. </li><li> 페트리 접시를 14mm 유리 바닥 사용을 판직접 200 ㎕의 최대 보유하고있는 내부 아니라 상에 1 × 10 5 대 식세포의 농도는, 그래서 그림 4와 같이이 볼륨을 초과하지에주의하십시오. </li><li> 세포가 1 시간 동안 37 ° C의 배양기에서 접시를 배치하여 유리 페트리 접시에 부착 할 수 있습니다. </li><li> 500 U / ㎖에서 1 ㎎ / ㎖ 및 IFNg에서 LPS로 보충 미디어를 먹이의 1-2 ML을 추가하고 세포가 하룻밤 배양 할 수 있습니다. </li></ol> 차 쥐과 대 식세포와 C의 3 공동 배양 콕 쿠스 네오 포르 만 <ol><li> 실험의 날, 420 X g에서 5 분 동안 원심 분리하여 하룻밤 접종 문화와 펠렛 효모 세포를 1 ㎖를 제거합니다. </li><li> 위에서 언급 한 속도 및 시간에 다당류 glucuronoxylomannan (GXM)와 같은 매체 및 크립토 외 제품을 제거하기 위해 멸균 PBS로 세포를 3 회 반복한다. </li><li> 100 희석 : 멸균 PBS 1 ㎖에 재현 탁 된 세포 펠렛과 1을 확보. t의 피펫 10 μL그는 혈구에 희석 세포를 계산합니다. 하나의 MOI에서 효모 세포를 추가합니다 5. 1 × 105 식세포가있는 경우 예를 들면, / 웰, CN의 5 × 106 / ㎖의 세포 현탁액을 5 × 105 CN의 전량이 용액 100 ㎕를 추가한다. </li><li> 10 μg / ml의 농도에서 단클론 항체 18B7 항체와 함께 먹이 미디어의 1 ml의 크립토 세포 현탁액의 계산 된 볼륨을 추가합니다. 5 분 동안 실온에서 세포 현탁액을 배양한다. </li><li> 유리에서 공급 된 용지를 제거은 페트리 접시를 바닥 및 멸균 PBS로 1 배를 씻는다. 물론 직접 옵 소닌 CN의 100 μl를 추가합니다. </li><li> 세포가 10 % CO 2와 37 ° C에서 2 시간 동안 배양 할 수 있습니다. </li><li> 공동 배양 후, 대 식세포는 CN을 내면화 한 것을 현미경으로 확인합니다. 내면화 고려하기 위해, 크립토 셀은 명확하게 식세포의 범위 내에서 볼 수 있어야합니다. </li><li> 멸균 PBS로 세척 페트리 접시의 3 배는 모든 extracellula를 제거합니다R CN. </li><li> 1-2 단클론 항체 18B7의 추가없이 미디어를 공급 ml의 설정 현미경을 추가합니다. </li></ol> 디지털 광학 현미경을 활용하여 실시간으로 4 떠올리 비 용해성 세포 외 유출 주 :, CO 2 배달 및 가열 장치가 필요합니다 포함 된 첨부 배양 챔버와 함께 제공되는 현미경을이 실험을 수행 할 수 있습니다. <ol><li> 실험 전에 적어도 30 분에 37 ° C에 대한 배양 챔버를 미리 따뜻하게. CO 2 장치는 실험 시작시 5 %를 읽고 있는지 확인합니다. </li><li> 장소 유리 CO 2 뚜껑 커버, 더 열 탈출 없도록 모든 문을 닫고, 현미경 플랫폼에서 페트리 접시를 바닥. </li><li> 위상 콘트라스트 현미경으로 10X 대물 렌즈를 이용하여 감염된 대 식세포의 클리어 필드에 초점. 실험을 바탕으로 연구 될 필요가 얼마나 많은 대 식세포에 의해 사용 목적을 결정한다. </li><li> 타에 현미경 소프트웨어를 설정이미지에게 24 시간 동안 매 4 분 애. </li><li> 24 시간 후, 실험이 완성에 도달 한 것 및 361 화상의 총 수집 된 것이다. 5 % 압축에 .jpegs으로 이러한 이미지를 내 보냅니다. 이미지 J 소프트웨어를 사용하여 초당 5 프레임에서 비주얼 스택으로 이미지를 볼 수 있습니다. 동영상이 게시 또는 프리젠 테이션을 볼 수 할 필요가있는 경우, AVI로 또는 형식 뷰어에 가장 적합한에 따라 .MOV 파일 중 하나로 저장합니다. </li></ol>

<ol><li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryptococcal+Interactions+With+the+Host+Immune+System%5BTitle%5D)+AND+%22Eukaryotic+cell%22%5BJournal%5D)">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li>
<li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryptococcus+interactions+with+macrophages%3A+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen%5BTitle%5D)+AND+%22Cellular+microbiology%22%5BJournal%5D)">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li>
<li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans%5BTitle%5D)+AND+%22Future+microbiology%22%5BJournal%5D)">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li>
<li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+pathogenic+bacteria+and+fungi+%E2%80%94+a+case+of+convergent+evolution%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Intracellular pathogenic bacteria and fungi — a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li>
<li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages%5BTitle%5D)+AND+%22BMC+immunology%22%5BJournal%5D)">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16(2007).</li>
<li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+cell-to-cell+spread+of+a+pathogenic+yeast%5BTitle%5D)+AND+%22BMC+immunology%22%5BJournal%5D)">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15(2007).</li>
<li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li>
<li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expulsion+of+live+pathogenic+yeast+by+macrophages%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li>
<li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2%2F3+complex-mediated+actin+polymerisation%5BTitle%5D)+AND+%22PLoS+pathogens%22%5BJournal%5D)">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li>
<li>Carnell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li>
<li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans%5BTitle%5D)+AND+%22Infection+and+immunity%22%5BJournal%5D)">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li>
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</ol>

저자는 우리가 더 경쟁 재정적 이해 나 관심의 다른 충돌이 없음을 선언합니다.

여기서 우리는 곰팡이 식세포 작용을 기록하고 24 시간 동안 실시간으로 분석 할 수있는 방법을 설명 하였다. 본 연구실은 비 용균 세포 외 유출의 시간적 측면 많은 공부이 프로토콜을 사용하고이 프로세스를 둘러싸고 형태 요소를 확인하기 위해 실시간으로 현미경을 사용하는 것을 계속할 것이다. 이 기술은 이상적인 결과를 산출하기 위해서는 특별한주의가 프로토콜의 ?...

크립토 세포와 대식 세포 사이의 곰팡이 감염의 단계가 아니라 1-3 설명되어 있습니다. 효모 세포는 대 식세포에 의해 섭취 된 후, 상호 작용의 다양한 발생할 수 대식구는 세포 외 공간으로의 진균 부하 해제 용해되거나, 그것의 세포막 (4)의 범위 내에서 효모 세포를 유지하여 감염을 제어 할 수있다. 비 용균 세포 외 유출, 식세포가 주변 환경으로 크립토 셀의 일부 또는 전부를 소거하는 프로세스 또는 인접 셀 및 호스트와 병원체 모두 5 가능한 남아 : 그러나, 몇 년 전에 새로운 결과 두 그룹에 의해 독립적으로 설명 된 -8. 몇몇 연구는 이러한 상호 작용의 뒤에 분자 메커니즘을 이해하려고했지만 우리는 여전히이 현상을 원동력에 대한 전체 이해를 필요가 없습니다. 기능은 실시간으로 영상을 캡쳐하고 그 후 여러 감염을 분석 할에드 대 식세포는 하나가 곰팡이 감염시 형태와 대 식세포 심지어 스트레스에서 타이밍, 공간 능력, 변화에 관해서 비 용해성 세포 외 유출을 둘러싼 물리적 특성에 대해 많은 질문에 대답 할 수 있습니다. 세포 배양기의 것과 필적 환경에 수납 될 수 있도록함으로써, 우리는 식세포 진균 세포 상호 작용의 동적 특성을 엿볼 수있다. 연구진은이 과정의 다양한 구성 요소를 연구하기 위해이 기술을 사용하고 있습니다. 또 다른 그룹은 비 용해성 세포 외 유출이 단백질 도메인을 핵 WASH 10 삭제 있었 세포에서 차단 된 것을 보여주기 위해이 방법을 사용하는 동안이 과정에서 phagosome의 역할은, 형광 염색과 액틴 차단제 구를 사용하여 분명했습니다. 사이토 카인 시그널링의 효과도 실시간 현미경 (11)을 사용하여 확인되었다. 이 프로세스는 C. 한정되지 않는다 그와 같은 콕 쿠스 네오 포르 만은 칸디다 알비 칸스 (Candida albicans)로 관찰되었다, anothe대 식세포 (12)을 감염시키는 능력을 가진 곰팡이 병원균 r에. 이러한 연구 결과는이 방법이 우리에 대해 너무 조금 알고있는 지역에 풍부한 정보를 제공 할 수있는 방법에 대한 예입니다.

<table><tbody><tr><td>Sabouraud Dextrose Agar</td><td>BD</td><td>DF0109-17-1</td><td></td></tr><tr><td>Sabouraud Dextrose Broth</td><td>BD</td><td>DF0382-17-9</td><td></td></tr><tr><td>Dubelcco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Corning Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Atlanta Biologicals</td><td>S12450</td><td></td></tr><tr><td>NCTC 109</td><td>Invitrogen</td><td>21340-039</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>HEPES Buffer</td><td>Corning Cellgro</td><td>25-060-Cl</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Corning Cellgro</td><td>25-025-Cl</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td>Toxic</td></tr><tr><td>3003 Tissue Culture Petri Dishes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>08772E</td><td></td></tr><tr><td>CellStripper</td><td>Corning Cellgro</td><td>25-056-Cl</td><td></td></tr><tr><td>Mat-tek Glass Bottom Culture Dishes</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L3137</td><td></td></tr><tr><td>Mouse IFN-g</td><td>Roche</td><td>NC 9222016</td><td></td></tr><tr><td>mAb 18B7</td><td>Non-commerical antibody produced in our lab</td><td></td><td></td></tr><tr><td>Axiovert 200M Microscope with Incubating Chamber</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

크립토 콕 쿠스 네오 포르 만에 의한 대 식세포의 감염의 많은 측면을 광범위하게 연구하고 잘 정의되어 있습니다. 그러나, 명확하게 이해되지 않는 한 특정의 상호 작용은 세포 외 유출 용균 비이다. 이 과정에서 효모 세포는 대 식세포와 CN 가능한 모두 나뭇잎 제대로 이해 메커니즘에 의해 세포 외 공간으로 해제됩니다. 여기서 우리는 시간 경과 현미경에 의해 24 시간 감염 기간에 대해 개별적으로 감염된 대 식세포의 큰 숫자를 수행하는 방법에 대해 설명합니다. 감염된 대 식세포가 세포 배양 인큐베이터과 동일한 조건을 제공 현미경에 부착 된 CO 2 분위기의 가열 챔버 내에 수용된다. 라이브 디지털 현미경 정적 이미지에서 사용할 수없는 호스트와 병원체 간의 동적 상호 작용에 대한 정보를 제공 할 수있다. 그 반대의 경우도 마찬가지 대식 세포가 곰팡이 감염을 처리하는 방법에 대한 단서를 제공 할 수 각 감염된 세포를 시각화 할 수 있다는. 이 기술의 전복잡한 현상 뒤에있는 역학을 공부 SA 강력한 도구입니다.

이 기술은 이미지를 생성 세포 C. phagocytosed 후에 발생하는 주변 환경 정보를 수집하기 위해 다양한 방식으로 분석 될 수있다 콕 쿠스 네오 포르 만이.도 1에 도시 된 바와 같이, 감염되거나 감염되지 않은 아르 중 약 100 대 식세포있다. 이 대 식세포의 각각은 시청자에게 매우 미세한 수준에서 호스트 병원체 상호 작용을 연구 할 수있는 기회를 제공합니다. 그...

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

우리는 macrophage- C.를 시각화하는 방법에 대해 설명합니다 콕 쿠스 네오 포르 만 스 (CN) 디지털 광 현미경을 사용하여 비 - 용해성 세포 외 유출의 프로세스에 특정 중점을 실시간으로 상호 작용. 개별적 감염된 대 식세포를이 방법을 사용하는 것은 이러한 현상의 다양한 양태를 확인하기 위해 조사 될 수있다.

의 비 용해성 세포 외 유출을 시각화<em&gt; 크립토 콕 쿠스 네오 포르</em&gt; 디지털 광학 현미경을 사용하여 대 식세포에서

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

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JoVE Journal

Immunology and Infection

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

11.8K 조회수.  Albert Einstein College of Medicine.  우리는 macrophage- C.를 시각화하는 방법에 대해 설명합니다 콕 쿠스 네오 포르 만 스 (CN) 디지털 광 현미경을 사용하여 비 - 용해성 세포 외 유출의 프로세스에 특정 중점을 실시간으로 상호 작용. 개별적 감염된 대 식세포를이 방법을 사용하는 것은 이러한 현상의 다양한 양태를 확인하기 위해 조사 될 수있다. 

비디오: Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

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 ...

연구

면역학 및 감염병학

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

숙주 세포와 관련하여 병원성 곰팡이의 정량화를 위해 이미지 계측법의 사용

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

공 초점 현미경으로 고립 된 폐포 대 식세포의 반대로 곰팡이 활동을 평가

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

대 식세포 콜레스테롤 고갈과의 식균 작용에 미치는 영향<em&gt; 크립토 콕 쿠스 네오 포르</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

마우스 대 식 세포에 의해 식 균 작용의 시간 경과 3D 영상

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

크립토코커스-아메바 상호 작용 을 연구할 때 현미경 기술과 형광 읽기의 상호 보완적 사용

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

제브라피시 배아에서 사체내 현미경 검사를 통해 진균 감염 시 대식세포 리액세포 사멸 시 시각화

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

인간 단핵구 유래 대식세포에서 근접성을 유도한 염증성 카스파제의 시각화

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

탐식의 초기 단계를 시각화

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

생물학

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

<meta charset="utf8"></head><body>Live Fluorescence, Inverse Imaging of Cell Ruffling 및 Macropinocytosis

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Live Fluorescence, Inverse Imaging of Cell Ruffling 및 Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

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