우리는 폴라 Cobos 및 실험실 지원 박사 브로스 Vassiliou을 감사하고 각각 마우스 대 식세포을 제공한다. 우리는 또한 지침 연구실 매니저 리처드 Criasia을 감사하고 재료를 도와줍니다. 마지막으로, 저자는 비디오 제작에 도움을 Ramanpreet의 우르 감사합니다.

1. 조직 문화 <ol><li> 콜레라 독소 결정에 대한 뮤스 musculus 대 식세포 (BALB / C 마우스)를 사용합니다. 처음에는 문화 M.의 공급 업체의 지침에 따라 musculus 세포. </li><li> 10 % 태아 소 혈청 완료 L-글루타민과 Dulbecco의 수정 이글의 중간에 BALB / C 마우스 세포를 전파하고, 1 % 항진균 / 항생제 또는 RPMI 1640 기본 매체는 10 % 태아 소 혈청, 5 %의 L-글루타민, 1로 완료 % 항진균 / 항생제 시약. </li><li> 37 환기 캡 75cm 2 코닝 플라스크에 세포를 성장 ° C, 95 % 공기와 5 % CO 2. </li><li> 지방산 결정의 결정을위한 기준, 제조업체의 지침으로 배달 24 시간 이내에 인체 장 상피 세포를 가져온다. </li><li> 37 10 % FBS 및 30 NG / ML 인간의 EGF와 함께 완료 HybriCare 미디어에서 배출 캡 ° C.와 75cm 2 코닝 플라스크에 인간의 장내 상피 세포를 전파 항생제를 사용하지 마십시오제조 업체의 지침에 따라 / 항진균 시약. </li><li> 자랄 때 약 70 %로 모든 셀 (1:3) 분할합니다. </li><li> 동결 표준 냉동 프로토콜을 사용하여 연구를 통하여 모든 셀을 가져옵니다. </li><li> 참고 : 지방산의 큰 규모의 농도 측정의 경우, 초기에 빠른 성장, 유지하게 쉬운 마우스 대 식세포를 사용합니다. 미세 규모의 지방산 농도 측정을 위해, 인간의 상피 세포를 사용합니다. 모든 콜레라 독소 치료 (설명 참조) 마우스 대 식세포를 사용합니다. </li></ol> 2. 지방산과 콜레라 독소 치료 <ol><li> 에서, 올레산 리놀레산과 리놀렌산을 전송 회사에서 제공하는 멸균 유리 병에 유리 앰플. </li><li> 별도의 멸균 에펜 도르프 튜브에 각각의 지방산을 전송 및 1:6 희석에 100 % 에탄올에 먼저 녹여 다음 10 ㎍ / μL의 최종 농도에 대한 RPMI 1640 완전 미디어합니다. </li><li> 와동 각 솔루션 및 TRANSF어 그것을 냉장고에 보관 유리 병 (-20 ° C)합니다. </li><li> 2,500 세포에서 / 잘 96 잘 조직 배양 플레이트에 플레이트 세포. </li><li> MTT 분석을위한 준비 지방산 세포 치료에 200 μL에 총 잘 볼륨을 가지고 적절한 전체 미디어를 추가 할 수 있습니다. </li><li> 24 시간 증식 기간이 지난 후에 용지를 제거하고 새로운 용지로 교체합니다. </li><li> (참조 결과) 우물 테스트 할 각 지방산의 적절한 농도를 추가합니다. 200 μL에 총 잘 볼륨을 가지고 전체 미디어를 추가 할 수 있습니다. </li><li> MTT 분석을 시작하기 전에 24 시간 동안 처리 한 번호판을 품어. </li><li> cryovials 1 MG / ML 및 나누어지는 솔루션의 농도로 PBS에서 콜레라 독소를 녹이고 2-8 cryovials를 저장 ° C. </li><li> 세포 치료의 경우, 1 NG / μL의 최종 작업 농도도 멸균 PBS (산도 7.4) 또는 불완전한 DMEM에 독소 1:100 희석. </li><li> 콜레라 독소와 세포를 치료하려면위에서 설명한대로 MTT 분석, 플레이트 세포를위한 준비. </li><li> 24 시간 증식 기간이 지나면 지방산 응용 프로그램 (위)에 사용되는 절차에 따라 우물을 테스트 할 콜레라 독소의 적절한 농도를 (우리의 농도에 대한 참조 결과)를 추가합니다. </li><li> MTT 분석을 시작하기 전에 24 시간 동안 처리 한 번호판을 품어. </li><li> 양 (양의 용지에 걸쳐 C)와 지방산 및 콜레라 독소 트리트먼트 서비스, 완전 배지 및 70 % 에탄올로 세포를 품어 각각 모두 대조군 (음수 C)와 같은. </li><li> 모든 샘플 및 치료는 양성 대조군에 대한 더 많은 복제 (본 연구에서는 N = 6)와 세 개의 복제 (N = 3)의 최소 사용하십시오. </li></ol> 3. MTT 분석 실험 <ol><li> 2.5 MG / ML의 농도에 M​​TT 원액을 만든다. </li><li> 테스트 할 96 잘 플레이트의 각 웰에 용액을 제거하고 신선한 완벽한 미디어 솔루션을 200 μL로 교체합니다. </li><li> 광고각각의 well에 MTT 용액 D 10 μL. </li><li> 37 ° C 3 ~ 4 시간 동안 접시를 품어. </li><li> 미디어 솔루션을 폐기하고 각 well에 이소프로판올 0.04 M의 HCl 100 μL를 추가합니다. </li><li> 5 분 동안 실온에서 접시를 품어. </li><li> 각 우물에서 새로운 원심 분리 관에 대한 해결책을 전송합니다. </li><li> 1 분 또는 펠릿이 형성 될 때까지 상온에서 20,000 XG에 원심 분리기. </li><li> 마이크로 플레이트 리더를 각 샘플의 20-40 μL간에 데이터를 전송합니다. </li><li> 분광 광도계를 사용하여 570 nm에서 흡광도를 참조하십시오. </li></ol>

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이 연구의 저자는 본 연구의 결과로부터 재정 관심이 없다. 본 연구를위한 기금은 킨 대학 FT에 제공되었지만, FT는 현재 Kingsborough 커뮤니티 칼리지에서 직원입니다.

지방산의 농도와 콜레라 독소의 제안 지방산 점막 면역을 강화하는 방법의 정확한 메커니즘은 알 수 있지만, 몇몇 연구들은 유익한 효과를 조사하기 위해 시도했습니다. 우리의 연구는 세포가 세포가 세포의 생존에 큰 영향없이 견딜 수있는뿐만 아니라 콜레라 독소의 최대 농도가 견딜 수있는 지방산의 최대 농도를 결정하는 방법을 제공하는 것을 목표로하고있다. <p class="...

같은 올레산, 리놀레산과 리놀렌산 등의 지방산의 건강 혜택은되고 문서화되는 것을 계속했다. 예를 들어, 올레인산은 몸 1,2 지용성 약물의 침투를 용이하게하는 데 도움이 포화 지방산 3를 대체 할 때 24 %가 관상 동맥 심장 질환을 감소시키고, X-연결되어 같은 Adrenoleukodystrophy 4와 같은 신진 대사 질환을 치료하는 데 사용됩니다 지방산 대사의 유전 질환. . 포유류의 아라키돈 산, 리놀산 (올레인산과는 달리)에 대한 필요한 전구체가 신체에 의해 합성되지 않는 등 아마씨 소비와 같은 외부 소스를 통해 얻을 수 있어야하지만 5 연구에서는 다음과 같은 리놀레산 여러 유익한 건강 효과를 보여 피부를위한 안티 에이징 속성, 6 항 염증, 대장 암과 전립선 암 세포의 7 감소 확산, 8 및 비만을 싸우는 능력 증진 오F 심장 혈관 건강. 9 리놀렌산 치주 염증, 10, 변조 트롬 복산과 프로 스타 사이클린의 생합성을 감소하는 역할을한다. 11 Arpita 12 V.에 담즙 지방산의 영향과 콜레스테롤을 공부 독성 요인과 운동의 콜레라의 식입니다. 야마자키 13 빨강 칠리 페 퍼, 기타 자연적으로 추출 된 화합물의 메탄올 추출물이 잠재적 인 콜레라 독소의 생산을 줄일 수 있다고 지적했다. 그것은 점막 면역을 제공을 통해 콜레라​​와 같은 전염성 질병의 예방과 퇴치를 위의 지방산 (예 : 아마 씨앗 등)이 풍부한 식품의 사용을 고려하는 것이 생각할 수있다. 우리는 세포 증식 분석, 인간 장 상피 세포가 세륨에 해로운 영향없이 견딜 수있는 지방산의 최대 농도를 사용하여 지방산을 용해하고 결정하기 위해 조사를 실시LL 생존. 우리는 올레산, 리놀레산과 리놀렌산은 낮은 농도에서 세포 생존에 유익한 효과를 제공하는 가설을하지만, 높은 농도에서 그들은 세포에 독성이 될 것입니다. 우리는 또한 콜레라 독소를 용해하고 BALB / C 마우스 대 식세포는 세포 생존에 큰 감소없이 견딜 수있는 콜레라 독소의 최대 농도를 결정 하였다. 우리는 심지어 매우 낮은 수준에서 세포 생존에 콜레라 독소의 독성 효과를 가설. 콜레라 독소를 가용화 및 세포 생존에 큰 감소없이 견딜 수있는 독소의 최대 크기를 결정하기 위해 그것을 사용하는 방법은 미래 연구에 대한 이점을 제공합니다. 예를 들어, 위의 방법의 조합은 지방산 콜레라 감염에 대한 점막 면역과 세포를 제공하는지 여부를 확인하는 데 사용할 수 있습니다. 우리가 아는 한,이 합리적이고 방법론은 탐구되지 않았습니다. 우리는 디우리의 예비 데이터는 올레산, 리놀레산과 리놀렌산은 콜레라 감염에 대한 점막 면역과 세포를 제공하는지 확인하기 위해 나중에 조사에서 사용할 수있는 방법을 scuss.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells/Reagent</td>
		 </tr>
		 <tr>
			<td>Mus musculus macrophages</td>
			<td>ATCC</td>
			<td>ATCC RAW 264.7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle's Medium</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal bovine serum </td>
			<td>Bio-west</td>
			<td>S0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotic/antimycotic </td>
			<td>Hyclone</td>
			<td>SV3007901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human intestinal epithelial cells </td>
			<td>ATCC</td>
			<td>ATTC CCL-241</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HybriCare media </td>
			<td>ATCC</td>
			<td>46-X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Oleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-1376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L-2376</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>BD Falcon 96-Well Cell Culture Plates</td>
			<td>BD Biosciences </td>
			<td>351172</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer with Dynex Revelations 4.22 software</td>
			<td>Dynex</td>
			<td>91000101</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and balb/c mouse macrophages

예방 및 비 - 인간과 인간의 질병의 완화 지방산의 긍정적 인 역할을하고 광범위하게 문서화되는 것을 계속했다. 이러한 역할은 염증의 예방뿐만 아니라 전염성 질병에 점막 면역 등의 전염성 및 비 전염성 질환에 영향을 이용하실 수 있습니다. 콜레라 박테리아 비브리오 콜레라에 의한 급성 장 질환이다. 그것은 개발 도상국에서 발생하고 방치하면 사망을 초래할 수 있습니다. 콜레라 백신은 존재하지만, 그들은 항상 효과적이지 다른 예방 방법이 필요합니다. 우리는 각각 마우스 BALB / C 대식 세포와 인간의 창자 상피 세포를 사용하여 허용 세 가지 지방산의 농도 (올레산, 리놀레산과 리놀렌산)과 콜레라 독소를 결정하기 위해 밖으로 설정합니다. 우리는 농도 범위와 NO 지방산의 특정 농도를 결정하기 위 지방산과 사용 세포 증식 분석을 용해인간의 장 상피 세포의 생존에 해로운 t. 우리는 콜레라 독소를 용해와 농도 범위와 통계적으로 BALB / C 대식 세포의 세포 생존 능력을 감소하지 않는 콜레라 독소의 특정 농도를 결정하기 위해 분석에 사용됩니다. 우리는 최적의 지방산 농도 사이에 1-5 μL / NG, 그 처리 기준 &lt;30 ng를 할 수 콜레라 독소합니다.이 발견 이 데이터는 예방이나 콜레라 감염의 완화 지방산에 대한 보호 점막 역할을 발견하는 것을 목표로 미래의 연구에 도움이 될 수 있습니다.

지방산의 최적 농도의 결정 지방산의 최적 농도는 세포의 성장에 필적하거나 결과 상대적으로 낮은 변동성 제어 세포의를 초과하는 최대 농도로 정의됩니다. 올레의 최적 농도를 결정하기 위해, 리놀레산과 리놀렌산 세포가 처음 큰 단위로 각 지방산의 농도를 변화하고 나중에 작은 단위로 처리 하였다. 그림 1은 세포의 생존을 사용하여 치료에 대한 긍정적 인 제...

Watch this Scientific Journal Video about Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages at JoVE.com

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

우리는 크게 악영향 지방산과 독소를 가용화 및 세포 생존 분석에서 그들을 사용하여 세포의 생존에 영향을 미치지 않았다 개의 지방산 (올레산 리놀레산과 리놀렌산)과 콜레라 독소의 허용 농도를 결정하기 위해 밖으로 설정합니다.

인간의 장내 상피 세포와 BALB / C 마우스 대 식세포를 사용하여 웬만한 지방산과 콜레라 독소 농도의 결정

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

determination-tolerable-fatty-acids-cholera-toxin-concentrations

Research

JoVE Journal

Immunology and Infection

9.9K 조회수.  Kingsborough Community College. 우리는 크게 악영향 지방산과 독소를 가용화 및 세포 생존 분석에서 그들을 사용하여 세포의 생존에 영향을 미치지 않았다 개의 지방산 (올레산 리놀레산과 리놀렌산)과 콜레라 독소의 허용 농도를 결정하기 위해 밖으로 설정합니다.

비디오: Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

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

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

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

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

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

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

연구

면역학 및 감염병학

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

마우스 소장에서 돌기 세포 및 Macrophages의 분리 및 특성화

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

장 상피 세포의 장내 세균의 침입<em&gt; 체외</em&gt; 크게 수직 확산 챔버 모델을 사용하여 향상

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

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

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

마우스 대 식세포의 타고난 면역 반응을 조사하기 위해 RNA 간섭을 사용하여

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

포유류 세포의 박테리아 감염 후 스트레스 과립 형성의 면역 형광 분석

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

포화 지방산은 세 라마 이드 관련 된 대 식 세포 세포 죽음을 유도

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

중생의 연구에 대 한 기본 위 상피 단층 문화 마우스 및 인간의 파생

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

병원성 메커니즘 연구를위한 BALB / c 마우스에서 약물 유도,자가 면역 간염의 유도

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

인간 유도 만능 줄기 세포 유래 장 소기관을 사용 하 여 살 모 넬 라 및 기타 병원 균에 대 한 상피 세포 보호 연구 및 수정

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...