이 작품은 자연 과학 및 캐나다의 공학 연구위원회에서 보조금에 의해 지원되었다 (NSERC, 더 298515-2011을 부여하지 않음). AL은 박사 학위를받는 사람입니다 자연 과학 및 캐나다 (NSERC)의 공학 연구 협의회 및 MS에서 장학금 (, 아니 부여합니다. MOP123246 CIHR) 건강 연구의 캐나다 연구소에서 교부금에서 지원되었다.

모든 절차는 센터 드 공들인 뒤 센터 Hospitalier 드 난 몬트리올 대학교의 기관 동물 관리위원회 (CRCHUM)의 승인 후 동물 관리 지침에 캐나다 문화원에 따라 수행 하였다. 1. 격리, 식별, 문화 쥐 복막 대 식세포의 <ol><li> 3.8 %의 맥주 티오 글리콜 레이트 매체를 준비합니다. 이렇게 증류수 1,000 mL에 티오 글리콜 레이트 배지 38g을 일시 중단합니다. 완전히 매체를 용해 끓여 솔루션을 가져와. 15 분 동안 121 ° C 오토 클레이브에 의해 멸균. RT 18, 어둠 3 개월까지 보관합니다. 참고 : 탁도가 세균 오염을 나타내는 개발하는 경우 폐기하십시오. 무균 유지 경우 용액을 어둠에서 1 년 동안 유지 될 수있다. </li><li> 각각의 양해 각서 (MOU)의 복강에 3.8 % 브루 티오 글리콜 레이트 배지 1 ㎖를 주입, 23 G 바늘에 부착 된 1 ml를 주사기를 사용하여SE는 3 일 기다려. 각 마우스의 새로운 주사기와 바늘을 사용합니다. </li><li> 나트륨 펜토 바르 비탈 (100 ㎎ / ㎏)을 복강 내 주사하여 마우스를 마취하고 경부 탈구에 의해 마우스를 안락사. 호흡을 확인하여 적절한 마취를 확인합니다. 일반적으로 빠른 호흡은 마우스가 깊이 마취되어 있지 않음을 나타냅니다. </li><li> 70 % 에탄올로 각 마우스의 복부를 씻으십시오. 가위를 사용하여, 복막의 하부 중앙선을 따라 횡 절개를 수행한다. </li><li> 집게를 사용하여 투명 복막 피부를 노출 복부 피부를 뒤로 당겨. 20 G 주사 바늘에 연결된 5 ㎖ 주사기를 사용하여, 각각의 마우스의 복강 내로 감기 DPBS 5 ㎖를 주입. </li><li> 복강에 부드러운 마사지를 수행하고있는 기관을 천공하지 않고 신중하게 유체를 대기음. 바늘을 제거하고 50 ML 원뿔 원심 분리기 튜브로 복강 액을 분배. </li><li> 냉장 원심 분리기 400 XG에 10 분 동안 원심 분리기.세포는 전체 과정 동안 차가운 유지한다. RPMI 배지 1640 뜨는에 resuspend 세포 펠렛을 폐기하십시오. </li><li> 혈구를 사용하여 세포를 계수하고 1 × 106 세포 / ml의 세포 밀도로 조정한다. </li><li> F4 / 80과 antibodie 대해 마우스 당 1 × 106 세포를 사용하여 유동 세포 계측법에 의해 분리 된 세포의 표현형을 특성화 (표면 항원은 대 식세포에서 발현). </li></ol> 2. 세포 치료 <ol><li> 직접, 차단 후, 각 웰에 1 × 10 6 세포를 추가합니다. 37 ℃에서 1 시간 2 그들을 배양하여 6 웰 플레이트에 부착 쥐의 복강 대 식세포를 남겨주세요. 부드럽게 따뜻한 PBS로 3 회 세척하여 비 부착 세포를 제거합니다. </li><li> 이어서, 900 μL의 무 혈청 DMEM에서 배양 세포 다음 TLR 리간드의 존재하에 24 시간 동안 : (Pam3CSK4-TLR1 / 2 (0.5 ㎎ / ㎖), 폴리 (I : C) -TLR3 (10 ㎎ / ㎖) ; LPS-TLR4 (100 NG / ㎖), 편모 - TLR5 (100 NG / ㎖) FSLTLR6 1 / 2 (100 NG / ㎖); ssRNA40-TLR7 (1 μg의 / ㎖); ODN1826-TLR9 (1 μM). 참고 : 각 리간드 10 배 재고 솔루션을 준비합니다. 따라서 10 배 희석을 수행, 각 웰에 후자의 100 μl를 추가합니다. </li></ol> 3. R​​NA 분리 <ol><li> 피펫을 통해 여러 번 각 웰에 1 ml의 트리 졸을 첨가하고, 세포 용 해물을 전달하여 6 웰 플레이트에서 직접 모든 매체를 Lyse 세포를 제거한다. 핵 단백질 복합체의 완전한 분리를 허용하도록, RT에서 5 내지 10 분 동안 균질화 된 샘플을 인큐베이션. </li><li> 1.5 ml의에 해물을 전송 RNA 분해 효소와 DNase의 무료 마이크로 원심 튜브. 1 ㎖의 트리 졸 0.2 ml의 클로로포름을 추가합니다. 15 초 동안 손으로 적극적으로 튜브를 흔들 5 ~ 10 분 동안 실온에서 그들을 품어. 4 ℃에서 15 분 동안 12,000 XG에 원심 분리기. 낮은 붉은 단계 (페놀 - 클로로포름)로 분리 혼합물, 계면 무색 위 수성 상을 관찰한다. RNA는 수성 상에 독점적으로 남아있다. </li><li> 트란계면을 방해하지 않고 새로운 튜브에 sfer 조심스럽게 위 수상. 이소 프로필 알콜 0.5 ㎖로 혼합하여 그것에서 RNA를 침전. 4 ℃에서 10 분 동안 12,000 XG에서 10 분간 원심 분리하고 실온에서 샘플을 인큐베이션. RNA는 튜브의 측면 및 저면에 흰 석출물 펠렛을 형성. </li><li> 상층 액을 제거합니다. 75 % 에탄올 1 ㎖로 한 번 RNA 펠렛을 씻으십시오. 4 ℃에서 5 분 동안 7,500 XG에 텍싱 및 원심 분리하여 샘플을 섞는다. 간단히 RNA (10 분 동안 건조 공기)을 건조. 0.1 ml의 DEPC (RNAse가없는 물)에서 RNA를 녹이고 55 ℃에서 10 분 동안 품어. </li><li> 분광 광도계를 사용하여 RNA를 정량화. 이렇게하려면 샘플을 DEPC 물에 100 배 희석하고 260 nm의 파장에서 읽을. RNA 농도는 것입니다 : 외경 260 × 40 NG / UL X 희석 계수를. </li><li> RNA 농도를 균일화하고 첫 번째 가닥의 cDNA 합성 키트에 대한 RT-PCR 시스템을 사용하여 제조업체의 지침에 따라 cDNA를 합성. 으로제조자의 지침에 따라, 실시간 PCR 실시간 DNA 검출 시스템 (45 회)에 의해 유전자의 mRNA 수준을 측정한다. 예 β 액틴 및 GAPDH 두 하우스 키핑 유전자와 유전자 발현 수준을 정상화. 참고 : ml의 500 μg의 /에 RNA 농도를 정상화 및 cDNA 합성 0.5 μg의를 사용합니다. </li></ol>

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The authors have no competing financial interests.

대 식세포는 생존을 위해 매우 중요하다 및 면역 학적 목표에 대한 호스트를 조작 할 수있는 유혹 대상을 제공합니다. TLRs를 인식하고 다른 분자의 발견은 면역 논쟁의 중심 대 식세포를 수행 하였다. 대 식세포는 사이토 카인, 손상 관련 분자 패턴 분자 (감쇠) (20)과 병원균 (PAMPs가) 21 그룹과 연관된 분자를 포함한 다양한 자극에 반응한다. 이러한 다양한 자극 응답은 대 식세포의 ...

세망 내피 식세포 시스템은 골수, 혈액, 간 및 비장 등의 다양한 조직 및 기관에서의 세포로 구성된다. 대 식세포는 광범위하게 그들은 특히 타고난에 참여하고 제어하는​​ 면역 반응과 명확한 감염을 적응 적 신체, 주위에 분포한다. 숙주 방어에서의 역할뿐만 아니라, 대 식세포는 상처 치유 및 조직 1,2- 항상성 유지에 중요한 역할을한다. 또한, 대 식세포는 면역 기능 만 중요한 것이 아니라 적극적으로 철 항상성 3에 참여한다. 체내에서 철의 약 80 %가 노화가 4 대 식세포에 의해 포식되고있는 경우에는, 적혈구 내의 헤모글로빈에 존재한다. 매일, 이러한 대 식세포는 적혈구 유래 철의 25 mg의 재활용 및 플라즈마 (5)에 자사의 전송을 제공한다. 또한, 감염 및 염증 동안 대 식세포 염증성 철 availabili 감소 혈청 철을 격리시키는모두 전신 및 지역 수준 6-8에서 병원균에 타이. 대 식세포 주로 간세포 철 대사 (9), (10)의 마스터 조절기 간주됩니다 hepcidin라는 항균 펩타이드를 생산하는 것이 아니라 연구는 보여 주었다. Hepcidin은 주로 염증 자극에 의해 증가 및 만성 염증 11 ~ 13시 대 식세포 철 격리 부분적으로 책임이있다. 대 식세포에 hepcidin 표현이 잘 이해되지 않습니다, 우리는이 규정에 수신자 같은 수용체의 가능한 역할 (TLR들)을 공부했다. TLR에 주로 대 식세포에서 발견되고 그들의 활성화에 중심적인 역할을한다. 또한, 간에서의 LPS 유도 hepcidin 식 TLR4 (13)에 의존한다. 따라서, 우리의 연구를 실행하도록, 우리는 뮤린 복막 마크로파지의 분리에 기초한 방법을 사용 하였다. 대식 세포주 대체로 대식 스터드에 이용된다이거; 그럼에도 연장 배양 유전자의 손실을 야기하고 이러한 세포주에서 면역 기능을 저하 할 수있다. 따라서, 복강 대 식세포에서의 분리는 매우 중요하다. 마우스 복강 대 식세포 13-15을 수확 할 수있는 이상적인 위치를 제공합니다. 고립 된 쥐의 복강 대 식세포는 면역 기능에 관한 여러 연구 편리합니다. 그러나, 복막의 대 식세포의 수가 방대한 연구 불충분하고, 마우스 당 약 1 × 106 식세포를 추정한다. 따라서, 대 식세포의 출력을 높이기 위해, 예컨대 티오 글리콜 레이트와 같은 멸균 제는 유도 세포 수확 직전 복강 내로 주사 하였다. 티오 글리콜 레이트 주사 후, 마우스 당 식세포의 수율은 10 배 증가 하였다. 대 식세포 수율, 대 식세포의 모집, 엄마의 결과, 염증 반응을 유도하는 자극으로 맥주의 티오 글리콜 레이트 배지 행위의 증가에도 불구하고그러나 Y 불필요 유전자 발현에 영향을 미친다. 따라서, 비 처리 된 대 식세포로 구성된 대조군이 각각의 실험에 포함되어야한다. 우리의 손에, 높은 염증에 의해 자극 hepcidin 표현은 복막 대 식세포를 이끌어 비 처리 티오 글리콜 레이트 검출되지 않았다. 또한, 연구는 맥주의 티오 글리콜 레이트가 많은 대 식세포를 모집하지만 그들에게 (16)을 활성화하지 않는 것으로 나타났습니다. 한편, 맥주의 티오 글리콜 레이트는 대 식세포는 리소좀 효소의 증가하지만 섭취 미생물을 죽이는 17의 감소를 유발 하였다. 비 - 유도 대 식세포와 비교할 때 16 그러나, 탐식 능력은 영향을받지 않았다. 접시에서 배양하면, 복막 마크로파지 따라서 복강으로부터 분리 된 세포의 다른 유형에서 자신의 분리를 허용 부착된다. 이어서, 절연 식세포 다른 TLR에 작용 제로 감염시켰다.마지막의 mRNA는 배양 된 세포로부터 추출하고, 유전자 발현은 정량적 역전사 - 중합 효소 연쇄 반응 (QRT-PCR)을 이용하여 분석 하였다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td>
			<td>475</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycollate</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>19032-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td>
			<td>WISENT INC Canada (QC)</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td>
			<td>WISENT INC Canada (QC)</td>
			<td>350-000-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 and 5 ml syringes</td>
			<td>BD USA (NJ)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>20g and 23g needles</td>
			<td>BD USA (NJ)</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes placed on ice</td>
			<td>Sarstedt (Newton, MA, USA)</td>
			<td>62.547.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cells Lysis Buffer</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>R7757-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80 antibody</td>
			<td>BIO-RAD ( CA, USA)</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32 antibodies</td>
			<td>Pharmingen, {Mississauga, ON, CA)</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>Axygen Scietific (CA,USA)</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific (ON, Canada)</td>
			<td>UN1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>JT Baker (PA, USA)</td>
			<td>70566</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol (in DEPC treated water)</td>
			<td>Commercial Alchohols (QC, Canada)</td>
			<td>17394</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>216.542.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript RT-PCR system</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor Gene 3000</td>
			<td>Montreal Biotech, (Kirkland, QC, Canada)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect SYBR Green I PCR kits</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>204141</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

감염과 염증 동안, 순환 단핵 세포는 혈류를 떠나 그들이 대 식세포로 분화 조직으로 이동한다. 마크로파지는 미생물의 광범위한 발전을 통해 보존 된 분자 패턴을 인식 표면 수신자 같은 수용체 (TLR들)을 표현한다. TLR에 보통 유전자 발현의 변화와 연관된 대 식세포의 활성화에 중요한 역할을 담당한다. 대 식세포는 많은 질병에 중요하며 치료를위한 매력적인 대상으로 등장했다. 다음의 프로토콜에서는 맥주의 매체를 이용하여 티오 글리콜 레이트 뮤린 복막 대 식세포를 분리하는 과정을 설명한다. 후자는 이에 따라이 10 배 식세포 수율을 올릴 것이다, 복막에 단핵 세포 마이그레이션을 높일 것입니다. 몇몇 연구는 골수, 비장 또는 복막 대 식세포 유래를 사용하여 수행되었다. 그러나, 복강 대 식세포는 분리에 따라 더 성숙한 것으로 나타났다과 기능에 더 안정되었다성만 및 표현형. 따라서, 쥐의 복강에서 분리 한 대 식세포는 다른 면역 및 대사 연구에서 봉사 할 수있는 중요한 세포 집단을 제시한다. 격리되면 대 식세포는 다른 TLR 리간드로 자극하고, 결과적으로 유전자 발현을 평가 하였다.

우리는 제 1 유동 세포 계측법에 의해 고립 된 쥐의 복강 대 식세포를 특징. 이렇게하려면, 우리는 구체적 만 대 식세포에 의해 표현 마커를 인식 (F4 / 80) 항체를 사용 하였다. 이 특성은 절연 식세포의 백분율을 결정하고, 분리 과정 중 얻어진 세포와 구별 할 필요가있다. (도 1)에 도시 된 바와 같이, 세포의 비율은 F4 / 80이 지속적으로 95 % 초과 인 것으로 밝혀졌다 항원을 발현. 이어서,...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

쥐 복막 대 식세포의 분리는 수신자 같은 수용체 자극하면 유전자 발현 분석을 수행하기

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.0K 조회수.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

비디오: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

마우스 복막 구멍 세포의 분리

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

연구

면역학 및 감염병학

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

수신자 같은 수용체 활성화 레굴라팅에 Neu1 Sialidase 활동 감지

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Monocytes과 인간 혈액의 작은 샘플에서 돌기 세포의 리니지 특정 수신자 같은 수용체 - 매개 시그널링의 정량 이미징

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

쥐과 잇몸 세포의 분리, 처리 및 분석

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

사용<em&gt; 시겔 flexneri</em&gt; 자식 작용 - 세포 뼈대의 상호 작용을 연구하는

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

레지오넬라 뉴모 필라 외부 막 소포 : 분리 및 대 식세포에 그들의 프로 - 염증 가능성 분석

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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. ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

사용 하 여 계통 발생 분석은 진 핵 유전자 원산지 조사

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Murine 골 복 막 대 식 세포 활성화에 대 한 항 체 기반 약물 효과의 평가

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...