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><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&amp;rsquo;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>6-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&amp;ndash;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 &amp;mu;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 &amp;mu;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>20 G and 23 G 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>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. ...

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JoVE Journal

Immunology and Infection

36.2K 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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

성인 간세포의 혼합 차 문화에서 Macrophages을 분리하는 간단하고 효율적인 방법

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

투과성 멤브레인 지지대를 이용한 공동 배양을 이용한 대식세포 활성화에 대한 종양 분비 파라크린 리간드의 효과 연구

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

암 연구학

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 ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

인간의 두 그라데이션 원심 분리하여 단핵구와 테프론 코팅 세포 배양 가방에서 대 식세포로 분화의 분리

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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 ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Toll-like Receptor Signaling의 억제제에 대한 식세포 면역 세포에서의 생체 활성 나노 입자의 스크리닝

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

의학

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

대 식 세포에의 한 사 멸 티 모 세포 침 몰의 실험 분석

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

복막 대식세포(Peritoneal Macrophage)에 의한 Apoptotic Thymocytes의 Efferocytosis 분석

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...