이 작품은 연구 그랜트 그랜트 - - 에이드 일본의 농림 수산 장관의 식품 나노기술 프로젝트에서.에 의해 투자되었다

1. 간의 재관류 <ol><li> 나트륨 pentobarbital 용액 (50 MG 나트륨 pentobarbital / kg 체중)의 intraperitoneal 주사로, 성인 남자 스프 라그 - 돌리 쥐 (몸 무게 1백50-2백그램 주 된 10-15에서) 마취. </li><li> 스테인리스 냄비에 동물을 장소와 복부 및 흉부에 70 % 에탄올을 스프레이. </li><li> 피부에서 작은 잘라 껍질을 확인하십시오. 가위로 절단하여 복부를 엽니다. </li><li> 포털 정맥의 위치를​​ 확인 포탈 정맥과 느슨하게 클럼프 아래 수술 스레드를 전달합니다. </li><li> 클럼프 하부 베나의 정맥과 간으로 혈액 역류를 방지하기 위해 정맥 mesenteric의 말초 부분을. 가위로 다이어프램을 절단하여 흉강을 열고 심장을 노출. </li><li> 포털 정맥에 플라스틱 카테 테르 (2mm 직경)를 삽입하고 단단히 카테터 주변의 스레드를 조입니다. </li><li> 37 prewarmed ° C.; 세척 용액 (pH는 7.2 칼슘 2 + - MG 2 + 0.5 MM EGTA, 10 MM의 HEPES 및 4.2 MM NaHCO 3가 포함된 무료 HBSS)이 탑재되는 재관류 시스템에 카테터를 연결 </li><li> 10 ML / 분 속도로 현장에서 재관류를 시작합니다. 동시에, 오른쪽 아트리움 벽에 상처를합니다. 10 분 재관류를 계속합니다. </li><li> collagenase 솔루션 재관류 솔루션을 스위치 [유형 보충 10mM HEPES 및 4.2mM NaHCO 3가 포함된 HBSS I collagenase (0.05 %)과 트립신 억제제 (50 μg / ML), 산도 7.5] 10, 그리고 perfuse - 속도로 20 분 10 ML / 분 간 약간 부풀 및 장액의 막 아래에있는 간 구조의 붕괴에 대한 기호를 표시하기 전까지. </li></ol> 2. Parenchymal Hepatocyte 풍부한 세포 분획의 준비 <ol><li> 제거하고 신중하게 collagenase 솔루션 25 ML을 포함하는 멸균 비커에 간 전송. 가위로 작은 조각으로 말하다. </li><li> 결과 세포 현탁액에 감기 멤의 75 ML을 추가합니다. 부드러운 pipetting 후, 여과수는 셀 스트레이너 (100 μm의)를 통해 결합 조직 및 소화되지 않은 조직 파편을 제거합니다. </li><li> 4 ° C (브레이크 해제)에서 1 분 50 XG 50 ML 원뿔 튜브와 원심 분리기에 여과물을 전송합니다. </li><li> 조심스럽게 뜨는 폐기하십시오. 감기 멤와 펠렛을 Resuspend. </li><li> )을 세 번 2.4 반복합니다. </li></ol> 3. 간세포의 혼합 차 문화 <ol><li> DMEM가에 100 μm의 β - 메르 캅 토 에탄올, 10 μg / ML 인슐린, 100 μg / ML 스트렙토 마이신과 100 U / ML 페니실린, 그리고 씨앗과 보충 10% 열을 inactivated FCS를 포함 구성된 성장 매체 2.5에서 얻어진 세포)를 일시 중지 6.7 X 10 4 세포 / cm 2 (중간 5 X 10 6 세포 / 술병 / 12 -15 ML)의 밀도에 : 5-10 조직 문화 flasks (75cm이 표면적). </li><li> 문화에게 flasksat 37 품어 ° 5% CO 2 분위기에서 C - 95 %의 공기. </li><li> 성장 매체마다 2~3일 간격을 바꾸십시오. </li></ol> 4. Macrophages의 선택적 분리 <ol><li> 문화 12 일 후에, macrophages의 세포 시트에 분아 따위에 의해 번식. 이러한 세포가 80 문화 flasks의 흔들어 상호에 의해 정지 아르 - 30 분 분당 120 스트로크를 37 ° C. </li><li> 100mm 이외의 조직 문화 학년 플라스틱 접시에 배지를 전송합니다. 한 플라스틱 접시 두개의 T75 flasks에서 매체를 풀. 성장 매체와 함께 리필 문화 flasks는 배양기로 돌아갑니다. </li><li> 37 30 분 플라스틱 요리를 품어 ° 5 % CO 2 분위기에서 C - 95 %의 공기. 다른 오염 세포 안 반면 Macrophages은 쉽게이 부화 과정에 따라 이외의 조직 문화 등급 플라스틱 요리에 첨부합니다. </li><li> 매체 대기음 부드럽게 PBS로 플라스틱 접시를 씻어. 이 단계를 세 번 반복합니다. </li><li> 그릇에 TrypLE 익스프레스 솔루션 1 ML을 추가하고 37 10 분에 대해 품어 ° 5 % CO 2 분위기에서 C - 95 %의 공기. </li><li> 성장 매체 9 ML을 추가합니다. 부드럽게 세포 스크레이퍼에 의해 연결된 macrophages를 다쳤어요. </li><li> ° C (브레이크)를 25 5 분 180 XG에 15 ML 원뿔 관과 원심 분리기에 세포 현탁액을 전송합니다. </li><li> 뜨는을 취소하고 성장 매체 1 ML를 추가합니다. 활발한 pipetting하여 단일 세포로 분열시키다. hemocytometer - 계산하여 휴대폰 번호를 열거합니다. </li><li> 절연 단계는 두 개 이상의 주 2~3일 간격으로 같은 문화 flasks와 반복 수 있습니다. </li><li> 절연 macrophages는 3.1에서 설명한 성장 매체 교양 수 있습니다. </li></ol> 5. 대표 결과 성인 쥐의 간 세포의 혼합 차 문화의 예제는 그림 2에 표시됩니다. Parenchymal hepatocytes 문화에 몇 일 이내에 상피 세포의 형태를 잃고, 타락한 또는 fibroblast 같은 세포로 변환.그렇다면, 하루 종일 6-12, 위상은 반대로 밝은 둥근 대식 세포와 같은 세포가 vigourouly fibroblastic 세포 시트에 분아 따위에 의해 번식. 문화 flasks의 흔들림으로, macrophages는 즉시 문화 매체에 정지하고 있으며, 플라스틱 요리 후속 전송과 부화는 macrophages의 선택적 유착 (그림 3)의 결과. 대식 세포와 같은 세포는 일 8로 일찍 수확하고, 숫자 12-14일에 최대한의 수준에 도달하실 수 있습니다. 이상 10 6 세포는 10 7 T75 당 문화 플라스크 (그림 3) 플라스크 당 총 세포 수율을 활성화, 2 주 이상 2 ~ 3 일 간격으로 반복 T75 문화 플라스크에서 수확 수 있습니다. 절연 macrophages의 purities은 같은 ED - 1 (그림 4), ED - 3, OX - 41 (그림 4)와 Iba1 9.로서 쥐 대식 세포 특정 항체와 flowcytometry 또는 immunocytochemistry에 의해 평가 95~99% 였는데 고립된 세포는 polystylene 비즈의 적극적인 phagocytosis (그림 5), 재조합 GM - CSF, LPS로 자극하고, multinucleated 거대한 세포 구 형성에 염증 / 안티 - 염증성 크린 시토킨의 분비에 proliferative 응답으로 macrophages의 전형적인 특성을 보여줍니다. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="그림 1" /> 그림 1. 쥐의 간 세포의 혼합 차 문화에서 대식 세포와 같은 세포의 선택적 분리 및 정화를위한 계획. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="그림 2" /> 그림 2. 쥐의 간 세포와 대식 세포와 같은 세포의 확산의 주요 문화. 화살표 multinuclear 거대한 세포를 나타냅니다. 스케일 바 = 100 μm의. Elsevier의 허가를 면역 방법의 논문집, Vol.360, 47-55 (2010) 9 Reprinted. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="그림 3" /> 그림 3. 떨고 첨부 방법으로 대식 세포와 같은 세포의 선택적 분리. 세포는 flasks를 흔들어하여 문화 매체에 정지 이후이 아닌 조직 문화 학년 플라스틱 접시에 양도하고, 37 ° C.에 incubated했다 도금 후 빠르면 10 분, 기타 오염 fibroblastic 세포가 정지 유지하면서, 접시 표면에 부착된 대식 세포와 같은 세포. PBS로 rinsing 후, 매우 정제된 세포 대식 세포의 인구는 얻은 것입니다. 이러한 세포는 문화의 40 분 이후에 전형적인 대식 세포의 형태를 얻고, 그리고 mitotic 세포는 자주 (화살표) 관찰되었다. 다른 문화의 시대에 flasks에서 발견한 세포 숫자의 후속 변경 내용이 표시됩니다. 값은 3 ~ 5 flasks에 평균 ± SD 있습니다. 실험은 세 번 반복되었으며 데이터를 다른 기호로 표시됩니다. 스케일 바 = 100 μm의. Elsevier의 허가를 면역 방법의 논문집, Vol.360, 47-55 (2010) 9 Reprinted. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="그림 4" /> 그림 4. 쥐의 단클론 항체에 대한 macrophages와 대식 세포와 같은 세포 Immunocytochemical 염색법. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="그림 5" /> 그림 5. 대식 세포와 같은 세포 FITC - 라벨 microbeads의 Phagocytosis.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

모든 동물은 동물 실험에 대한 기관의위원회에 의해 명시된 지침과 규정에 따라, 보관 및 동물 보건 국립 연구소에있는 동물 시설에서 운영되었다.

여기, 우리는 성인 쥐의 간 세포의 혼합 차 문화에서 macrophages를 얻을 간단하고 효율적인 방법을보고합니다. 우리의 방법은 쥐 간 세포의 혼합 문화 macrophages의 소설 proliferative 활동에 첫번째 의존하고 macrophages 9으로서의 생물 학적 특성을 바탕으로 이러한 세포의 후속 분리 및 정화. 그것은 성인 간 세포의 혼합 차 문화의 확산 macrophages는 Kupffer 또는 parenchymal hepatocytes 분율로 오염과 관련된 세포에서 유래있을 수도 있습니다. macrophages는 혼합 차 문화에 parenchymal hepatocyte 10 기타 fibroblastic 세포의 변화로 인해 특정 환경 변화에 대응 할 수도 있습니다. 결론적으로, 우리의 방법은 정교한 장비와 기술을 사용하여 쥐의 간 cellswithout의 주요 문화 충분한 번호 및 순도의 대식 세포와 같은 세포의 격리를 제공합니다. 또한, 절연 절차는 두 개 이상의 주 같은 문화 플라스크와 반복 수 있습니다. 이 방법은 다른 포유 동물 종에 적용할 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 나트륨 pentobarbital 용액 </td><td> 교리쓰 Seiyaku </td><td> Somnopentyl 사출 </td><td></td></tr><tr><td> 행크의 균형 소금 용액 (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> 에틸렌 글리콜 tetraacetic 산 (EGTA) </td><td> 시그마 - 알드리치 </td><td> E - 4378 </td><td></td></tr><tr><td> Collagenase </td><td> 와코 순수 화학 공업 (주) </td><td> 032-10534 </td><td> 세포 분리 학년 (롯 확인 필요) </td></tr><tr><td> 트립신 억제제 </td><td> 시그마 - 알드리치 </td><td> T - 9128 </td><td></td></tr><tr><td> 이글의 최소 필수 중간 (멤) </td><td> 시그마 - 알드리치 </td><td> M - 4655 </td><td></td></tr><tr><td> Dulbecco이 수정의 이글의 중간 (DMEM) </td><td> 시그마 - 알드리치 </td><td> D - 6459 </td><td> 포도당 - 타입 하이 </td></tr><tr><td> 소태아 혈청 (FCS) </td><td> HyClone </td><td> SH30070.03 </td><td> 열 ° C 30 분 56 inactivated. (롯 확인 필요) </td></tr><tr><td> TrypLE 익스프레스 </td><td> Invitrogen </td><td> 12,605 </td><td></td></tr><tr><td> Dulbecco의 인산염은 버퍼링 호수 (PBS) </td><td> Invitrogen </td><td> 14,190 </td><td> CA 2 + - MG 2 + 무료 </td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 시그마 - 알드리치 </td><td> M - 7522 </td><td> 주식 : 증류수 100 MM </td></tr><tr><td> 인슐린 </td><td> 시그마 - 알드리치 </td><td> I - 5500 </td><td> 주식 : 0.1N HCL 10 MG / ML </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Invitrogen </td><td> 15,070 </td><td></td></tr><tr><td> 셀 스트레이너 </td><td> BD Biosciences </td><td> 352360 </td><td> 메쉬 크기 : 100 μm의 </td></tr><tr><td> 조직 문화 플라스크 </td><td> 스미토모 Bakelite (주) </td><td> MS - 21250 </td><td> 표면적 : 75cm 2 </td></tr><tr><td> 비 - 조직 문화 플라스틱 요리 </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> 셀 스크레이퍼 </td><td> 코닝 주식 회사 </td><td> 3010 </td><td></td></tr><tr><td> 상호 흔드는 </td><td> TAITEC </td><td> NR - 1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Kupffer 세포가 간 특정 주민 macrophages이며, 1-3 간의 생리와 병리 학적 기능에 중요한 역할을한다. 간 macrophages의 절연 방법 4-6 잘 설명하고 있지만, 이러한 방법의 대부분은 이러한 원심 elutriator 및 기술 능력 등 정교한 장비가 필요합니다. 여기, 우리는 간략하게 구조 그림 1에 설명된 성인 쥐의 간 세포의 혼합 차 문화에서 충분한 번호 및 순도에 간 macrophages을 얻기 위해 소설 방법을 제공합니다. 2 단계 재관류 방법 7,8하여 간 세포의 분리 후, 대부분 parenchymal hepatocytes 구성된 분수가 준비하고 DMEM와 10 %의 FCS.Parenchymal hepatocytes 구성된 문화 매체와 T75 조직 문화 flasks에 씨앗을 품고 것은 상피 세포의 형태를 잃게 문화의 몇 일 이내에, 타락한 또는 fibroblast 같은 세포 (그림 2)로 변환. 로 문화의 날 6 주위 진행, 위상 대조 - 밝고 둥근 대식 세포와 같은 세포 (그림 2) fibroblastic 세포 시트에 분아 따위에 의해 번식을 시작합니다. 대식 세포와 같은 세포의 성장은 술병의 표면에있는 세포 시트를 덮고, 계속 일 1,200 최대 수준에 도달. 문화 flasks의 흔들림으로, macrophages는 즉시 문화 매체로 중단됩니다. 와 같은 다른 오염 세포가 정지 상태 macrophages의 선택 접착에 플라스틱 접시 결과의 후속 전송 및 짧은 부화 (그림 3). PBS 여러 rinses 후, 첨부된 macrophages은 수확하고 있습니다. 이상 10 6 세포는 두 개 이상의 주 (그림 3) 2-3일 간격으로 동일한 T75 조직 문화 플라스크에서 반복적으로 수확 수 있습니다.하거나 유동세포계측법에 의해 평가로 격리 macrophages의 purities은 95~99%되었습니다 쥐 대식 세포 특정 항체 (그림 4)와 immunocytochemistry가. 고립된 세포는 LPS로 자극하고, 형성시 polystylene 비즈의 적극적인 phagocytosis (그림 5), 재조합 GM - CSF에 proliferative 반응, 염증 / 안티 - 염증성 크린 시토킨의 분비를 보여 multinucleated 거대 세포 9. 결론적으로, 우리는 방법이 다른 포유 동물 종에 적용있을 복잡한 장비 및 skills.This없이 충분한 번호 및 순도에 간 macrophages를 얻을 수있는 간단하고 효율적인 방법을 제공합니다.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

쥐의 간 세포의 주요 문화 macrophages를 얻을 소설 방법을 설명합니다. 이 방법은 플라스틱 요리 선택적 첨부하여 문화 flasks 및 정화의 흔들림 다음 문화 macrophages의 확산을 활용합니다. 이 기술은 효율적으로 복잡한 장비와 기술없이 간 macrophages을 제공합니다.

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 ...

a-simple-efficient-method-to-isolate-macrophages-from-mixed-primary

Research

JoVE Journal

Immunology and Infection

37.9K 조회수.  National Institute of Agrobiological Sciences, Tsukuba, Japan. 쥐의 간 세포의 주요 문화 macrophages를 얻을 소설 방법을 설명합니다. 이 방법은 플라스틱 요리 선택적 첨부하여 문화 flasks 및 정화의 흔들림 다음 문화 macrophages의 확산을 활용합니다. 이 기술은 효율적으로 복잡한 장비와 기술없이 간 macrophages을 제공합니다.

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

기본 인적 Keratinocytes에서 Organotypic 뗏목 문화의 생성

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

연구

면역학 및 감염병학

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

신생아 쥐 뇌 조직의 혼합 Glial 세포 배양의 기본 Microglia 격리

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

유동 세포 계측법에 의해 인간 호중구에 핵 팩터 활성화를 감지 할 간단하고 효율적인 방법

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Nucleofection에 의해 인간의 THP-1 대 식세포의 고효율 형질

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

마우스 수지상 세포 하위 집합의 분리를위한 효율적이고 높은 수율 방법

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

간단하고 효율적인 접근 방법은 돌연변이 백시 니아 바이러스 벡터를 구축하기

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

마우스 골수에서 생성 및 GM-CSF의 식별 파생 폐포와 같은 대 식세포와 수지상 세포

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

마우스에서 차 간세포 문화에 염증 반응의 유도

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

결막에서 세포를 분리하고 표현하기위한 비 침습적 방법

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK과 만성 활성 엡 스타인-바 바이러스 감염을 가진 환자에서 T 셀 라인을 설정 하는 효율적이 고 간단한 방법

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...