저자 E1E4 항체의 종류 선물 샐리 로버츠 (버밍엄 대학, 버밍햄 영국) 감사드립니다. 이 작품은 국립 암 연구소 (4R00CA137160-03)에서 교부금에 의해 지원되었다.

1. Organotypic 뗏목 문화를위한 준비 <ol><li> 금속 뗏목 격자가 훨씬 먼저 분화 과정을 방해할 수있는 찌꺼기를 제거하는 삼가의 크롬을 함유하는 제품 황산으로 처리됩니다. 한 시간 만이라도 황산이 들어있는 유리 비커에 잠그다 금속 격자는 다음 지속적으로 수돗물로 씻어서 하룻밤. 린스 하룻밤 후, 뗏목 격자가 이중으로 증류수에 3-5시간 위해 씻어서되어야합니다. </li><li> 보트에 대한 지원을 제공하기 위해 서로 동등한 거리에서 0.5 cm에 대한 뗏목 세 가지 측면을 구부. 금속 격자 그런 다음 autoclaved해야합니다. </li><li> 2.2 g NaHCO3 및 4.8 g의 Hepes을 추가하여 10X reconstitution 버퍼를 준비합니다. 0.05 M NaOH의 100 ML에 디졸브. 필터 소독 및 -20 ° C.에서 5 ML의 aliquots에 저장 </li><li> 나트륨 중탄산염없이 10X DMEM을 준비합니다. 필터 소독 및 -20 ° C.에서 5 ML의 aliquots에 저장 </li><li> 마우스 표피 성장 인자를 준비 : EGF의 100 MG와 B의 10 밀리그램을 디졸브SA 10 ML에서 각각 ddH 2 0를 deionized. EGF와 BSA를 결합 및 100 ML의 총 볼륨의 deionized ddH 2 0 80 ML에 추가합니다. 필터 소독, 나누어지는 (5 ML) 및 -20 ° C.에 저장 전자 매체의 각 리터에 대해 1 밀리그램 / ML EGF (최종 농도 5 NG / ML) 5 ML에 추가합니다. </li></ol> 2. 콜라겐 젤의 작성 <ol><li> 하나는 콜라겐 젤은 각 뗏목이 필요합니다. 콜라겐이 응고하지 않도록 얼음에 보관해야합니다. 추위 쥐 - 꼬리 콜라겐 유형 I (최종 농도 3-4 밀리그램 / ML), 0.3 ML 10X reconstitution 버퍼, 0.3 ML 10X DMEM and1-2 X 10 6 마우스의 2.4 ML : 각 콜라겐 젤은 구성된 콜라겐 믹스 3 ML이 필요합니다 피더 세포와 같은 3T3 J2 섬유아 세포. 쥐의 꼬리 콜라겐의 높은 농도 솔루션의 경우 올바른 작동 농도를 달성하기 위해 0.02 M 초산에 희석. </li><li> 당신은 콜라겐의 양을, 10X reconstit위한 마스터 믹스를 계산 후 필요합니다 콜라겐 젤 (보트)의 개수를 결정ution 버퍼, 10X DMEM과 J2 섬유아 세포가 필요합니다. 콜라겐의 점성으로 인해 3-4 여분 젤류 각 마스터 믹스에 충분히 포함합니다. </li><li> 콜라겐 젤을 준비하려면, J2 섬유아 세포를 trypsinize와 매체로 무력화. 50 ML 원뿔 유리병의 모든 섬유아 세포와 장소를 결합. 만들 수 보트의 수를 결정하는 세포를 세어보세요. 다시 말하지만, 1-2x10 6 섬유아 세포는 뗏목 당 따라서 콜라겐 젤별로 요구하고있다. 낮은 속도로 세포를 내려 봐. </li><li> 10X reconstitution 버퍼, 10X DMEM과 계산 보트의 수를위한 콜라겐의 적절한 금액 fibroblast 펠렛과 resuspend에서 매체를 기음. 너무 빨리 응고에서 젤을 방지하기 위해 콜라겐이 마​​지막에 추가합니다. 콜라겐이 추가되면 젤로 거품의 도입을 방지하기 위해 부드럽게 빠르게 섞는다지만. 겔은 올바른 산도 나타내는 붉은 오렌지 색상이어야합니다. 겔 혼합물이 너무 노란색 경우 필터 소독 1N의 안약 몇 가지 추가NaOH와 혼합 부드럽게 정확한 색상을 구하십시오. </li><li> 콜라겐 3 ML 추가 : fibroblast 혼합물을 6 아니라 문화 접시의 우물에. 콜라겐 혼합물은 거품을 발생하지 않도록 천천히 각 우물의 측면 아래로 pipetted해야합니다. 37 ° C 조직 문화 인큐베이터에 금속판을 놓고 30 분 동안 응고 수 있습니다. </li><li> 30 분 후에, 경화 콜라겐 젤 위쪽에 EGF와 전자 매체 3 ML을 추가하고 배양기에 플레이트를 반환합니다. 젤은 최소한 하루에 37 ° C에서 보관 아니고 넷 일 이내에 사용해야합니다. 최적의 결과는 부화 두 가지 일 후에 발생합니다. </li></ol> 3. 차별 화를위한 준비 Keratinocytes <ol><li> 낮은 통로 keratinocytes는 약 70 % confluency로 성장해야한다. 각 뗏목은 1-2x10 6 keratinocytes가 필요합니다. keratinocytes을 Trypsinize 및 EGF와 전자 매체로 무력화. 만들 수 보트의 수를 결정하는 세포를 세어보세요. 낮은 spe에서 세포를 다운 던가에드. 계산된 각 뗏목 문화에 대한 EGF와 전자 매체의 1 ML에 펠렛을 Resuspend. </li><li> 틸팅 6 잘 판에 의해 콜라겐 젤에서 매체를 기음. 각 겔에 대한 EGF와 함께 신선한 전자 매체는 2 ML을 추가합니다. 각 겔 들어, 조심스럽게 잘에 이미 존재 전자 매체의 2 ML에 resuspended keratinocytes 1 ML을 추가합니다. 다음 부드럽게 pipet keratinocytes을 더한 미디어의 두 ML하고 가을 드롭이 많다는 다시 젤 향해 보자. 떨고하거나 번호판을 출렁 이는 것은 콜라겐 겔 중 한쪽으로 세포의 분포가 불규칙이 생길 수 있습니다. 37 ° C 배양기에서 접시를 놓습니다. </li><li> 세포는 매일 전자 매체를 대체, confluency로 성장해야한다. 세포가 합류하는 경우 중간에 변경 후 노란색 언젠가 매체 변경됩니다. 이것은 보통 2~4일 소요됩니다. 매체가 하루 네에 의해 변경되지 않은 경우 다음 단계로 진행합니다. </li></ol> 4. 뗏목 문화 만들기 <ol><li> 멸균 금속 뗏목 격자를 배치 멸균 집게를 사용하여, 양측 내려가 보자, 10cm 접시에. 콜라겐 겔에서 매체를 기음. 잘의 측면에서 젤을 느슨하게하려면, 동작을 위아래를 사용하여 멸균 주걱으로 젤의 주위에 이동하십시오. 콜라겐 젤을 제거하려면 약간 접시를 기울이고 주걱의 아래를 삽입하여 리프트. 그리드와 젤 사이에 기포를 생성하지 않고 금속 격자에 콜라겐 젤 놓는다. 두 젤류는 각 격자에 둘 수 있습니다. </li><li> 공기 - 액체 인터페이스를 만들기 위해서는, 뗏목 격자가 미디어를 감동적 있도록 접시의 바닥에 천천히 EGF없이 전자 매체를 추가하지만, 콜라겐 젤이 아닙니다. 미디어 그리드에 구멍을 통과해서는 안됩니다. 이 균일한 분화를 차단하므로 그리드 아래에 거품을 발생하지 않도록 천천히 매체를 추가하십시오. 37 ° C에서 보트를 부화하고 공기 - 액체 인터페이스를 유지하고 매일 매체를 변경합니다. 십사일 후 보트를 수확. </li></ol> 5. 대표 결과 때 t그 프로토콜이 기본 keratinocytes에서 제대로 수행됩니다, 성형 수술은 피부의 다른 레이어는 증거들이 잘 차별 상피집니다. 이것은 같은 그림 1B에 나타난 hematoxylin 및 eosin (H &amp; E)의 뗏목 문화의 부분을 더럽히는 것으로 histological 시험을 통해 달성될. H &amp; E 염색법은 보트와 계충화하다와 차별화하는 세포의 능력에 바이러스 또는 항바이러스 화합물의 효과의 형태를 검사하는 것도 유용합니다. 예를 들어, HPV-불후의 기본 인간 keratinocytes (HFK-31)로 만든 보트가 특히 두꺼운 확산의 증가 속도와 차별화 세포 활성 유지하는 HPV 단백질의 능력을 반영, 정상적인 인간의 포피를 keratinocytes (HFK)로 만든 보트와 비교된다 세포주기의 (그림 1B) 10. 상피 전역의 핵 보유에이 결과, 정상 keratinocytes가의 붕괴가 발생, 분화에 따라 세포주기를 종료 반면,핵 봉투. 더 자세히 분화를 조사하려면, immunohistochemistry는 cytokeratins뿐만 아니라, involucrin, 그리고 filaggrin 포함하여 특정 분화 마커를 조사하기 위해 수행할 수 있습니다. 다른 cytokeratins는 분화 7의 특정 단계에서 표현됩니다. 그림 2에 표시된 바와 같이, cytokeratin 10 (K10)가 기초 레이어로 표현되지 않으며 오직 점진적으로 차별화 세포로 구성되어 suprabasal 레이어에서 발견된다. HPV 긍정적인 보트에서 K10의 표현이 다시 keratinocyte 분화에 HPV 단백질의 영향을 반영하는 정상 keratinocytes, 그에 비해 지연될 수 있습니다. Immunohistochemistry은 또한 바이러스 유전자의 표현 프로필을 검사하는 데 사용할 수 있습니다. 그림 3에 표시된 것은 표현 상피의 맨 위에 레이어로 제한됩니다 늦은 단백질은 HPV 단백질 E1 ^ E4의 표현을 검토 대표적인 염색법이다. 예상했던대로 아무 염색법 정상 huma에서 뗏목 문화에서 관찰되지 않습니다포피의 keratinocytes 그렇죠. <img alt="그림 1" src="/files/ftp_upload/3668/3668fig1.jpg" /> 그림 1. 기본 keratinocytes이나 상피 세포 라인에서 organotypic 뗏목 문화를 준비하기위한 방법의) 개요. 안정적으로 HPV-31 (HFK-31) episomes, 또는 정상적인 인간의 포피를 keratinocytes (HFK)에서을 유지하는 인간의 포피를 keratinocytes에서 발생 organotypic 뗏목 문화 B) Hematoxylin 및 eosin 염색법. 각각의 상피 층은 물론 콜라겐 플러그로서 식별됩니다. <img alt="그림 2" src="/files/ftp_upload/3668/3668fig2.jpg" /> 그림 2. Cytokeratin 10 (K10) 표현식은 상피의 suprabasal 레이어로 제한됩니다. Immunohistochemistry은 K10에 항체를 사용하여 HFK-31 세포뿐만 아니라 정상 HFKs에서 생성된 organotypic 뗏목 문화의 교차 부분에서 수행되었다. 세포의 DNA는 DAPI로 counterstained되었다. 이미지는 conf 파일을 사용 잡혔었지ocal 형광 현미경. 화살표는 상피의 기저 계층을 나타냅니다. <img alt="그림 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> 그림 3. HPV E1 ^ E4 단백질은 후반 바이러스 생활주기의 생산 단계에서 생산된다. Immunohistochemistry은 E1E4에 항체를 사용하여 HFK-31 세포뿐만 아니라 정상 HFKs에서 생성된 organotypic 뗏목 문화의 교차 부분에서 수행되었다. 세포의 DNA는 DAPI로 counterstained되었다. 이미지가 공촛점 형광 현미경을 사용하여 캡처했다. 화살표는 상피의 기저 계층을 나타냅니다.

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우리는 공개 할게 없다.

여기서 일반적으로 상피 분화를 연구하는 데 사용할 수있는 방법을 설명하지만, 또한 쉽게 바이러스성 pathogenesis뿐만 아니라 잠재적인 치료제의 효능을 연구하기 위해 적용할 수 있습니다. 많은 바이러스 herpesviruses와 마찬가지로 HPV의 경우처럼, 또는 바이러스 생활주기의 어느 시점에서 감염의 주 사이트로서 중 상피 세포를 타겟팅합니다. 숙주 세포의 상호 작용 : organotypic 뗏목 문화를 성장하는...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 쥐의 꼬리 콜라겐 타입 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354,236 </td></tr><tr><td> NaHC0 세없이 DMEM </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td> 시그마 </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391,338 </td></tr><tr><td> 스테인레스 금속 격자 </td><td> 윌리엄스와 기질 (주) </td><td> CR-03063-040-100-S </td></tr><tr><td> 전자 매체 11 </td><td></td><td></td></tr><tr><td> 표피 성장 인자 </td><td> BD Biosciences </td><td> 354,010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

organotypic 상피 뗏목 문화의 발달은 체외 시스템을 충실히 recapitulates 상피 분화에 효율적으로 연구자를 제공합니다. 이 시스템에 대한 많은 용도가 있습니다. 예를 들어, keratinocytes의 입체 organotypic 뗏목 문화를 성장하는 능력은 인간 papillomavirus (HPV) 1의 연구에 중요한 이정표가되었습니다. HPV의 라이프 사이클은 긴밀하게 편평 상피 2의 분화에 링크되어 있습니다. 같은 Organotypic 상피 뗏목 문화가 여기에 바이러스 생산 3,4,5 포함한 전체 papillomavirus 수명주기를 재현 보여주었다. 또한, 이러한 뗏목 문화가 HPV와 생체내 감염에 따라 관찰 비슷한 dysplastic 병변을 나타냅니다. 따라서이 시스템은 또한 상피 세포 암뿐만 아니라, 일반적으로 상피 세포 분화에 대한 약물의 효과를 연구하는 데 사용할 수 있습니다. 원래 Asselineau 및 Prunieras 개발한 <s6&gt; 백업 및 Kopan 외 7.에 의해 수정, organotypic 상피 뗏목 문화 시스템이 콜라겐으로 세포의 성장과 관련된 일반적인, 비교적 쉬운 문화 모델로 성숙 공기 - 액체 인터페이스 (그림 1A)에서 관리하고 플러그. 10-14일의 과정 동안, 전지 계충화하다과 차별화, 차별화 특정 cytokeratins을 생산하고 전체 두께 상피 형성. 수확 보트는뿐만 아니라 표준 분자 및 생화 학적 기법으로, histologically 검사를 할 수 있습니다. 이 문서에서는 기본 인간 keratinocytes에서 뗏목 문화의 생성을위한 방법을 설명합니다. 동일한 기술은 설립 상피 세포 라인과 함께 사용할 수 있으며, 쉽게 정상 또는 질병 biopsies 8 시부터 상피 조직을 사용하기 위해 적용할 수 있습니다. 많은 바이러스가 대상 중 피부 또는 점막 상피 자신의 replicative 수명주기의 일부로. 지난 몇 년 동안의 가능성은 organotypic 뗏목 예찬에게 사용바이러스 숙주 세포 상호 작용을 연구하는 방법으로서 ures는 여러 herpesviruses뿐만 아니라 adenoviruses, parvoviruses 및 poxviruses 9 보여줘왔다. Organotypic 뗏목 문화는 따라서 바이러스 pathogenesis를 조사하기 위해 적응하고, 영구 세포 라인에 cultivable없는 이들 바이러스 소설 항바이러스 대리인을 테스트하는 유일한 수단입니다 수 있습니다.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

<em&gt; 체외에서</em모방하는&gt; 방법<em&gt; 생체내에서</em&gt; 상피 분화가 설명되어 있습니다. 대부분의 바이러스는 바이러스 라이프 사이클의 일부로서 상피 세포를 타겟으로,이 방법은 바이러스를 검사하는 수단을 제공합니다 : 호스트 상호 작용을보다 밀접하게 발생하는 것이 비슷한 것을<em&gt; 생체내에서</em&gt;. 이 기술은 기본 keratinocytes 설립 세포 라인뿐만 아니라, 정상 또는 질병 생검 조직에서 사용할 수 있습니다.

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 ...

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Research

JoVE Journal

Immunology and Infection

19.0K 조회수.  University of North Carolina-Chapel Hill.  체외에서 방법 생체내에서 상피 분화가 설명되어 있습니다. 대부분의 바이러스는 바이러스 라이프 사이클의 일부로서 상피 세포를 타겟으로,이 방법은 바이러스를 검사하는 수단을 제공합니다 : 호스트 상호 작용을보다 밀접하게 발생하는 것이 비슷한 것을 생체내에서. 이 기술은 기본 keratinocytes 설립 세포 라인뿐?...

비디오: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

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 ...

연구

면역학 및 감염병학

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

정렬 된 불멸화 된 B 세포로부터 재조합 인간 IgG 단일 클론 항체의 생성

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

준비 및의 Organotypic 흉선 슬라이스 문화의 응용 프로그램

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

차세대 일렉트로 차 인간 Th17 세포 작은 RNA 형질

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...