우리는 원고의 비판적 읽기 박사 다이앤 Baronas - 로웰 감사합니다. 이 작품은 과학 씨 그랜트의 FAU 찰스 E. 슈미트 대학 KJ에 엘리슨 의료 재단에서 노화 장학금 지원

1. XLD (크 실리 톨 리신 데스 옥시 콜레이트) 한천 플레이트 XLD 한천 XLD 한천 플레이트에 검은 식민지로 나타납니다 살모넬라 균에 대한 선택 성장 매체입니다. 오염 될 우려가없는 경우에는, 정규 LB 플레이트가 치환 될 수있다. <ol><li> 5 ㎖의 탈 이온수에 5.5 g의 XLD 한천과에 resuspend를 달다. </li><li> 모든 한천 젖은 때까지 철저하게 섞는다. 모든 덩어리가 사라과 매체가 완전히 재현 탁 될 때까지 탈 이온수 95 ML을 추가합니다. </li><li> 완전히 (압력솥하지 않음) 용해 매체를 끓인다. </li><li> 50 ℃의 실온에서 매체를 냉각 </li><li> 각 95 x 15mm (직경 x 높이) 플레이트 (파라 필름으로 밀봉 플레이트가 1 달 동안 4 ° C에 저장할 수 있습니다)에 25 ㎖의 한천을 부어. </li></ol> 2. 선충류 성장 중간 (NGM)의 RNAi는 플레이트를 먹이는 C. 준비 엘레 NGM 플레이트는 이전에 설명 된 19. 다음 절차는 짧게 RNAi의 먹이 접시를 만들기 위해 NGM 미디어에 항생제 암피실린 및 RNAi의 화학 유도 이소 프로필 β-D-1-티오 갈 락토 피 라노 시드 (IPTG)를 추가 할 설명되어 있습니다. <ol><li> 3g의 NaCl 1 L 탈 이온수 2.5 g 박토 펩톤을 녹입니다. </li><li> 미디어에 17g의 박토 한천을 추가합니다. </li><li> 45 분 동안 미디어를 압력솥 수욕에서 50 ° C로 미디어를 냉각. </li><li> 1 ㎖ 콜레스테롤 (95 % 에탄올에 5 ㎎ / ㎖) 1 ㎖ 1 M 염화칼슘, 1 ㎖ 1 M 망초, 25 ㎖의 1 M 인산 칼륨 완충액 (pH 6.0) : 다음과 같은 솔루션을 추가합니다. 잘 섞는다. </li><li> 1 ㎖ 1 M IPTG 500 ㎕의 암피실린 (멸균 100 ㎎ / ㎖)를 추가합니다. </li><li> 잘 솔루션을 섞어 피펫 보조를 사용하여 멸균 절차에 따라 25 ㎖의 혈청 피펫 (60) X 15mm (직경 x 높이) 페트리 접시에 붓는다. 12 ML 한천 각 접시를 채우십시오. 플레이트는 최대 1 달 동안 4 ° C에 저장할 수 있습니다. </li></ol> autophagy에 필수적인 유전자는 BEC-1은 살모넬라 감염에 대한 방어의 숙주 유전자의 기능을 검사하는 예로서 사용된다. 실험 절차는도 1 및 표 1에 도시된다. 감염의 RNAi-처리 된 동물의 제조 프로토콜은 괄호 안에 각 실험 단계의 일로, 다음과 같다. <ol><li> BEC-1 RNAi의 먹이를 접종 및 100 ㎎ / ㎖ 암피실린 (1 일) 보충 2 ㎖ LB 배지에 -80 ° C 냉동 박테리아의 조각을 배치하여 빈 벡터 L4440 RNAi의 박테리아 먹이를 제어 할 수 있습니다. 신선한 RNAi의 세균을 가지고 전체 실험 기간 동안 일주일에 한 번이 단계를 반복합니다. 사용하지 않을 경우 4 ° C의 냉장고에 문화를 저장합니다. </li><li> RNAi의 접시에 하룻밤 RNAi의 세균 배양을 100 ㎖의 종자. 세 BEC-1 RNAi의 세 제어 빈 V를 준비RNAi의 판을 엑터. 37 ° C에서의 하룻밤 (2 일)에서 번호판을 품어. </li><li> 37 ° C의 배양기에서 RNAi의 플레이트를 제거하고 벤치에서 실온까지 냉각 할 수 있습니다. 잘 공급 L4 야생형 N2 자웅 동체를 들고 그들을의 RNAi-1 벡 빈 벡터 RNAi의 판을 제어하는 전송. 세중의 플레이트에 플레이트에 세 벌레를 놓습니다. 단계 3.2 (3 일)에 설명 된대로 같은 날, RNAi의 판을 준비합니다. </li><li> 3.3 단계에서 제조 된 신선한 해당 RNAi의 플레이트에 36-40 시간 및 전송 웜에 대한 20 ° C 배양기에서 벌레와 RNAi의 번호판을 품어. 웜이 전송 된 후, 64 시간 (4 일)에 20 °의 C 배양기에서 접시를 품어. </li></ol> 4. 감염 살모넬라 준비 <ol><li> 줄무늬 살모넬라 -80 ° C 냉동 1 XLD 한천 접시에 하룻밤 (5 일 37 ° C에서 접시를 품어 </st룽&gt;). </li><li> 잘 고립 된 검은 살모넬라 식민지를 선택하고 (6 일) 밤새 진탕 37 ° C에서 2 ㎖ LB 배지에 접종. </li><li> 1 C.에 씨 80 ㎖의 살모넬라 균 배양 물 엘레 60 X 15mm (직경 x 높이) NGM 한천 플레이트와 총 6 판을 준비합니다. 6 시간 동안 실온에서 번호판을 품어. 세균 배양 접시 (주 7)에 잔디를 건조 형성한다. </li></ol> 살모넬라 5.을 감염 RNAi의 처리 벌레 <ol><li> 이동 BEC-1 RNAi의 처리 및 살모넬라 판에 빈 벡터 RNAi의 처리 L4 N2의 자웅 동체 (3 단계에서 설정 벌레의 자손)을 제어 할 수 있습니다. 각 그룹에 대해 3 접시에 접시 당 40 벌레를 놓습니다. 48 시간 (주 7) 20 ° C에서 웜 번호판을 품어. </li><li> 단계 3.1 및 3.2 (일 7에 설명 된대로 신선한 RNAi의 접시의 한 세트를 준비하고 <stroNG&gt; 8 일). </li><li> 48 시간 감염 후, 단계 5.2에서 제조 된 해당 RNAi의 접시에 살모넬라 균에 감염된 웜을 전송하고 20 ° C (9 일)에 품어. </li></ol> 6. 생존 분석 <ol><li> 매일 벌레의 생존을 점수와 알을 낳는 시간 동안 신선한 해당 RNAi의 플레이트로 웜을 전송합니다. 단계 3.1 및 3.2에 설명 된대로 이전에 각 웜 전송에 신선한 RNAi의 접시 세트를 준비합니다. 부드럽게 끝 평평 백금 와이어 웜 몸 (머리, 중간 부분 및 꼬리)를 터치합니다. 웜 본체의 어떠한 움직임이 관찰되지 않은 경우 웜 죽은로서 획득된다. </li><li> 매일 웜 또는 매일의 생존을 점수와 벌레가 산란을 중단 한 후 일주일에 두 번 신선한 해당 RNAi의 플레이트로 웜을 전송합니다. </li><li> 모든 벌레가 죽은 후에, 하나의 데이터 세트로 세중 판에서 생존 데이터를 풀. 같은 그래프 패드 P 적절한 통계 소프트웨어에 각 그룹의 입력 생존 데이터생존 곡선을 생성하고 Kaplan-Meier 생존 분석을 수행 할 rism. 전체 실험의 결론을 확인하기 위해 한 번 이상 반복한다. </li></ol>

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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

C. elegans의는 영양원으로 세균을 먹는 간단한 유전자 모델 생물이다. 따라서, C. 사이의 상호 작용을 조사하기 위해 장내 병원체 정상 세균 음식을 대체하기 쉽다 엘레 및 선택 병원체. 여기 프로토콜은 살모넬라 균 감염과 C를 결합하여 설명 엘레 RNAi의 살모넬라 균 감염에 대한 방어의 호스트 유전자의 역할을 조사하는 치료를 먹이. 이전 감염 프로토콜은 <...

독립 생활 토양 선충 Caenorhabditis의 엘레 많은 생물학적 질문을 연구하는 데 사용하는 간단하고 유 전적으로 다루기 쉬운 모델 생물이다. C. 엘레 지배적 자기 시비 자웅 동체로 존재합니다. 남성은 자발적으로 배우자 형성 1,2 동안 X 염색체의 비 분리에 의해 생성됩니다. 풍부한 음식, C.의 존재 엘레 지속적으로 성인 네 애벌레 단계를 통해 개발. 온도는 C.에 영향을 elegans의 개발; 빠른 개발은 높은 온도에서 관찰된다. 실험실에서, C. 엘레 음식 1,2 시드 박테리아 대장균 (주 OP50)와 한천 플레이트에서 20 ° C의 표준 온도에서 배양. 지난 10 년, C. 엘레 호스트 병원체 상호 작용 3-5을 연구하는 무척추 생물로 떠오르고있다. 자연에서, C. 엘레는 nutrien으로 박테리아를 먹는다T 소스, 2. 정상 세균 실험실 음식, OP50은 쉽게 C. 사이의 상호 작용을 조사하기 위해 다른 병원체로 치환 될 수있다 엘레 및 선택 병원체. 이러한 조건에서, 대장은 감염의 기본 사이트이다. 실제로, 세균성 병원체의 넓은 범위는 치명적 C. 감염을 보였다 엘레 3-5. 그람 음성 박테리아 살모넬라 균은 전 세계적으로 6,7 인간 식중독을 일으키는 위장 병원체이다. C. 이 박테리아가 복제 및 지속적인 장내 감염 8-10 전시로 엘레는 살모넬라 티피 뮤 리움 (Salmonella typhimurium)에 대한 좋은 모델 호스트입니다. C. 엘레 참신한 모두를 식별하는 데 사용되었으며, 이전에 공지 된 살모넬라 병원성 11 요인. 흥미롭게도, C. elegans의 면역 체계가 성공적으로 살모넬라 균의 복제를 제한합니다. 그것은 그 저해에게 이전에보고 된autophagy에 유전자의 ition는 C. 증가 살모넬라 복제를 렌더링 엘레 감염된 웜 (10)의 조기 사망의 결과. (여기에 autophagy라고 함) Macroautophagy이 저하 12 리소좀에 배달 세포질과 세포 기관을 격리시키는하기 위해 세포 내 세포막의 재 배열을 포함하는 역동적 인 과정이다. 에 autophagy는 C.에서 살모넬라 복제를 제한하기 위해보고되었다 엘레와 포유류 10, 13에. C. elegans의 게놈 염기 서열 첫 번째 다세포 진핵 생물의 게놈이었다; 이는 RNAi의 치료 14-16에 응답한다. 또한,이 RNAi의 RNAi의이 (16, 17)를 먹이로 알려진 표적 유전자의 이중 가닥 RNA를 포함하는 박테리아를 섭취하도록 웜을 실시함으로써 효과적으로 투여 될 수있다. 전체 게놈 RNAi의 먹이 라이브러리는 게놈 전체의 RNAi는 16, 18 심사에 대해 생성되었습니다. 여기서, 살모넬라 균 감염 프로프로토 콜은 RNAi의이 테스트 C. 허용하도록 먹이와 결합 살모넬라 균 감염으로부터 보호하기 위해 자신의 능력에 대한 관심 elegans의 유전자.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">BP9723-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XLD agar</td>
			<td>EMD Chemicals</td>
			<td>1.05287.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Fisher</td>
			<td>DF0140-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Fisher</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Fisher</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Fisher</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td>Gold Biotechnology</td>
			<td>12481C50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Fisher&#160;</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 95 x 15mm</td>
			<td>Fisher</td>
			<td>FB0875714G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 60 x 15mm&#160;</td>
			<td>Fisher</td>
			<td>08-757-13A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Serological pipet</td>
			<td>Fisher</td>
			<td>13-668-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Express Pipet-Aid</td>
			<td>Fisher</td>
			<td>13-675-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ6000 shaking incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival</td>
			<td>I-36DL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

지난 10 년, C. 엘레은 그람 음성 박테리아 살모넬라 티피 뮤 리움 (Salmonella typhimurium)에 대한 숙주 방어를 포함하여, 호스트와 병원체 사이의 상호 작용을 연구하는 무척추 생물로 떠오르고있다. 살모넬라 균은 C.의 소장에서 지속적인 감염을 설정 엘레 감염된 동물의 조기 사망의 결과. 면역기구의 수는 C.에서 확인되었다 살모넬라 균 감염에 대해 방어하는 엘레. autophagy에, 진화 적으로 보존 된 리소좀의 분해 경로는 C에서 살모넬라 균의 복제를 제한하기 위해 표시되었습니다 엘레와 포유류. 여기서, 프로토콜은 C. 감염 설명 벌레가 인간의 살모넬라 균 감염과 유사한 제한된 시간에 대한 살모넬라 균에 노출 된 살모넬라 티피 뮤 리움 (Salmonella typhimurium)와 엘레. 살모넬라 균 감염은 크게 C.의 수명을 단축 엘레 &lt;/ EM&gt;. 예를 들어 필수 autophagy에 유전자 BEC-1을 사용하여, 우리는 C.으로이 감염 방법을 결합 엘레 RNAi의 접근법은 먹이와이 프로토콜은 C.의 기능을 검사하는 데 사용될 수 보였다 살모넬라 균 감염에 대한 방어 엘레 호스트 유전자. 이후 C. elegans의 전체 게놈 RNAi의 라이브러리를 사용할 수 있으며,이 프로토콜은 포괄적 C.에 대한 화면으로 가능하게한다 게놈 전체의 RNAi 라이브러리를 사용하여 살모넬라 균 등 장내 병원균에 대한 보호 elegans의 유전자.

20 ° C에서, 야생 형 N2 벌레의 평균 수명은. 살모넬라 균 감염이 크게 10.5 일 N2 웜의 평균 수명을 감소 십칠일 (그림 2A 및 표 2)는 (P = 0.0002, 로그 순위 검정) (그림 2A ). 만약 C. 엘레 유전자는 살모넬라 감염에 대한 방어에 중요한 역할을, 그것은 그것의 억제는 살모넬라 감염에 대한 감수성을 부여 할 것으로 예상된다. ?...

Watch this Scientific Journal Video about A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium at JoVE.com

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans의 호스트 병원체 상호 작용을 연구 할 수있는 새로운 유전자 모델로 떠오르고있다. 여기에서 우리는 C. 감염 프로토콜을 설명 살모넬라 감염에 대한 방어의 숙주 유전자의 역할을 조사하는 이중 가닥의 RNAi 간섭 기법과 결합 된 살모넬라 티피 뮤 리움과의 elegans.

감염 프로토콜<em&gt; 예쁜 꼬마 선충</em&gt;와<em&gt; 살모넬라 티피 뮤 리움 (Salmonella typhimurium)</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K 조회수.  Florida Atlantic University.  C. elegans의 호스트 병원체 상호 작용을 연구 할 수있는 새로운 유전자 모델로 떠오르고있다. 여기에서 우리는 C. 감염 프로토콜을 설명 살모넬라 감염에 대한 방어의 숙주 유전자의 역할을 조사하는 이중 가닥의 RNAi 간섭 기법과 결합 된 살모넬라 티피 뮤 리움과의 elegans. 

비디오: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

확인을 위해 Allelotyping PCR 살모넬라 enterica serovars Enteritidis, 해더, 하이 델베르크, 그리고 Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

연구

면역학 및 감염병학

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

표현형 높은 처리량 분석<em&gt; 살모넬라 엔테</em&gt; 티피 뮤 리움 협회, 침략, 그리고 대 식세포에서의 복제

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

살 모 넬 라 typhimurium의 절연-대 식 세포에서 Phagosomes를 포함 하

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

살 모 넬 라 종의 심사에 대 한 높은 처리 플랫폼 /Shigella 종

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

꼬마 선 충 모델에서 Aeromonas 감염의 병 인 및 독성 평가

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

마우스 종아리에 일관된 마사지와 같은 섭동을 적용하고 근육 내 압력 변화를 모니터링

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

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 ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

만성 살모넬라 감염 유도 장 섬유증

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

살모넬라 감염 조직 배양 세포에서 유전자 발현의 NF-θB 의존성 루시퍼라아제 활성화 및 정량화

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

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