Name: isolation, transfection, and culture of primary human monocytes
Uploaded: 2019-12-16T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

저자는 유세포 분석 분석을 제공하기 위한 환자 샘플 및 세포 면역학 물질 대사 코어를 제공하는 HIV 임상/종양 Biorepository Core에 감사드립니다. 이 프로젝트는 NIH P20GM121288 및 P30GM114732에 의해 투자되었습니다.

아래에 설명 된 모든 방법은 루이지애나 주립 대학 건강 과학 센터 뉴 올리언스 기관 검토 위원회에 의해 승인되었습니다. 모든 혈액은 통보된 동의를 얻은 후에 수집되었습니다.
참고: 전체 절차는 생물 안전 수준 2(BSL2) 시설에서 멸균 조건하에서 수행되므로 생물학적 물질을 처리하는 데 주의가 사용됩니다. 특히, 각 단계는 생물 안전 성 캐비닛 에서 멸균 기술을 사용하여 ?...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+M1/M2+polarization+dynamically+adapts+to+changes+in+cytokine+microenvironments+in+Cryptococcus+neoformans+infection.">Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection.</a> MBio. 4 (3), e00264 (2013).</li><li>Raggi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Human+Macrophage+M1-M2+Polarization+Balance+by+Hypoxia+and+the+Triggering+Receptor+Expressed+on+Myeloid+Cells-1.">Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1.</a> Frontiers in Immunology. 8, 1097 (2017).</li><li>Van Overmeire, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF+and+GM-CSF+Receptor+Signaling+Differentially+Regulate+Monocyte+Maturation+and+Macrophage+Polarization+in+the+Tumor+Microenvironment.">M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.</a> Cancer Research. 76 (1), 35-42 (2016).</li><li>Vogel, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+macrophage+polarization+in+vitro:+maturation+and+activation+methods+compared.">Human macrophage polarization in vitro: maturation and activation methods compared.</a> Immunobiology. 219 (9), 695-703 (2014).</li><li>Almeida, M., Cordero, M., Almeida, J., Orfao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+subsets+of+peripheral+blood+dendritic+cells+show+distinct+phenotypic+and+functional+abnormalities+in+HIV-1+infection.">Different subsets of peripheral blood dendritic cells show distinct phenotypic and functional abnormalities in HIV-1 infection.</a> AIDS. 19 (3), 261-271 (2005).</li><li>Ciesek, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+TRAIL-dependent+cytotoxicity+of+CD1c-positive+dendritic+cells+in+chronic+hepatitis+C+virus+infection.">Impaired TRAIL-dependent cytotoxicity of CD1c-positive dendritic cells in chronic hepatitis C virus infection.</a> Journal of Viral Hepatitis. 15 (3), 200-211 (2008).</li><li>Granelli-Piperno, A., Golebiowska, A., Trumpfheller, C., Siegal, F. 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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR8+regulation+of+LILRA3+in+monocytes+is+abrogated+in+human+immunodeficiency+virus+infection+and+correlates+to+CD4+counts+and+virus+loads.">TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads.</a> Retrovirology. 13, 15 (2016).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Impairment+of+Myeloid+Dendritic+Cells+during+Advanced+Stage+of+HIV-1+Infection:+Role+of+Factors+Regulating+Cytokine+Signaling.">Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling.</a> PLoS ONE. 10 (10), e0140852 (2015).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+negative+regulators+of+cytokine+signaling+during+chronic+HIV+disease+cause+functionally+exhausted+state+of+dendritic+cells.">Increased expression of negative regulators of cytokine signaling during chronic HIV disease cause functionally exhausted state of dendritic cells.</a> Cytokine. 91, 118-123 (2017).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Li, H., Jiang, T., Li, M. Q., Zheng, X. L., Zhao, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Regulation+of+Macrophages+Polarization+by+MicroRNAs.">Transcriptional Regulation of Macrophages Polarization by MicroRNAs.</a> Frontiers in Immunology. 9, 1175 (2018).</li><li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Analyses+of+MicroRNA+Profiling+in+Interleukin-27+Treated+Monocyte-Derived+Human+Dendritic+Cells+Using+Deep+Sequencing:+A+Pilot+Study.">Genome-Wide Analyses of MicroRNA Profiling in Interleukin-27 Treated Monocyte-Derived Human Dendritic Cells Using Deep Sequencing: A Pilot Study.</a> International Journal of Molecular Sciences. 18 (5), (2017).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+miR-126-5p+Enhances+the+Inflammatory+Responses+of+Monocytes+to+Lipopolysaccharide+Stimulation+by+Suppressing+Cylindromatosis+in+Chronic+HIV-1+Infection.">MicroRNA miR-126-5p Enhances the Inflammatory Responses of Monocytes to Lipopolysaccharide Stimulation by Suppressing Cylindromatosis in Chronic HIV-1 Infection.</a> Journal of Virology. 91 (10), (2017).</li><li>Lodge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+MicroRNAs-221+and+-222+Inhibit+HIV-1+Entry+in+Macrophages+by+Targeting+the+CD4+Viral+Receptor.">Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor.</a> Cell Reports. 21 (1), 141-153 (2017).</li><li>Ma, L., Shen, C. J., Cohen, E. A., Xiong, S. D., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA-1236+inhibits+HIV-1+infection+of+monocytes+by+repressing+translation+of+cellular+factor+VprBP.">miRNA-1236 inhibits HIV-1 infection of monocytes by repressing translation of cellular factor VprBP.</a> PLoS ONE. 9 (6), e99535 (2014).</li><li>Riess, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferons+Induce+Expression+of+SAMHD1+in+Monocytes+through+Down-regulation+of+miR-181a+and+miR-30a.">Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.</a> Journal of Biological Chemistry. 292 (1), 264-277 (2017).</li><li>Buchacher, T., Ohradanova-Repic, A., Stockinger, H., Fischer, M. 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제시된 프로토콜은 단핵구 및 대식세포를 공부하기 위한 모형으로 HIV 감염한 과목에서 1 차적인 세포의 사용을 보여줍니다. HIV+ cART를 겪고 있는 환자는 여러 해 동안 감염과 함께 살고 있으며 손상된 면역 체계와 관련된 다른 공동 감염을 가질 수도 있습니다. HIV 만성 감염의 존재에서 면역 조절을 연구하기 위해 세포는 환자로부터 직접 수확되었습니다. miRNA가 세포 발달 및 분화에 중요?...

인간 면역 결핍 바이러스-1 (HIV-1) 감염은 기회 감염 및 후천성 면역 결핍 증후군 (AIDS)으로 이어질 수있는 심각한 면역 기능 장애를 유발합니다. cART를 겪고 있는 HIV 감염 환자는 낮은 바이러스 하중 및 일반적인 CD4+ T 세포 수에 의해 특징지더라도, 면역 계통의 기능은 암 발전의 증가한 리스크에 연결되는 역기능 면역 반응으로 이끌어 내는 이 개별에서 손상될 수 있습니다1. cART에 HIV 환자에 있는 면역 기능 장애의 기계장치는 크게 알려지지 않은 남아 있습니다. 따라서 환자 유래 면역 세포를 특성화하고 생물학 및 기능을 조사하는 것이 현재 HIV 연구의 중요한 구성 요소입니다.
단핵구와 대식세포는 면역 반응의 주요 조절자이며 HIV 감염2,3,4,5에서근본적인 역할을 합니다. 본질적으로 이질성 및 플라스틱, 대식세포는 고전적으로 활성화(M1) 또는 대안적으로 활성화(M2)로 광범위하게 분류될 수 있다. 이러한 일반적인 분류는 실험 조건을 설정할 때 필요하지만, 대식세포의 편광 상태는다양한사이토카인6,7,8,9에의해 역전될 수 있다. 여러 연구가 단핵구와 수지상 세포에 대한 HIV 감염의 영향을 조사했지만, 단핵구 매개 반응의 분자 세부 사항은 크게 알려지지 않은6,7, 10,11,12,13,14,15,16,17,18,19. 면역 세포 조절 및 기능에 관여하는 인자 들 중에서, microRNAs (miRNAs), 유전자 발현 후 전사적으로 조절되는 짧은 비코딩 RNA는, 주요 세포 경로의 맥락에서 중요한 역할을 하는 것으로 나타났다 (즉, 성장, 분화, 개발, 및 세포 자멸)20. 이들 분자는대식세포(21)의기능적 편광을 지시하는 데 필수적인 전사 인자의 중요한 조절자로 기술되었다. cART를 겪고 있는 HIV 감염한 개별에서 단핵구에 있는 miRNAs의 잠재적인 역할은 조사되었습니다, 그러나 필드에 있는 진행은22,23,24,25,26를훨씬 더 필요로 합니다 . 이 논문에서는 HIV 에 감염된 환자 및 대조군으로부터 1차 인간 단핵구로 miRNAs 및 siRNAs를 트랜스펙트하는 최적화된 방법에 대해 설명합니다.
이 프로토콜은 시판되는 시약과 키트에 의존하며, 기술 절차의 연속성은 임상 샘플로 작업할 때 불필요한 실험 변수를 제거하는 데 도움이 됩니다. 그럼에도 불구하고, 상기 방법은 중요한 팁(즉, 플레이트에 대한 세포의 순착을 촉진하기 위해 무혈청 매체로 도금된 세포 의 수 또는 간략한 인큐베이션)를 제공한다. 또한, 이 프로토콜에 사용되는 편광 조건은 출판된 작업27,28,29에서파생된다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Stain Buffer</td>
			<td>BD Horizon</td>
			<td>563794</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD14 PerCP</td>
			<td>Invitrogen</td>
			<td>46-0149-42</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD163 BV711</td>
			<td>BD Horizon</td>
			<td>563889</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD209 BV421</td>
			<td>BD Horizon</td>
			<td>564127</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD80 FITC</td>
			<td>BD Horizon</td>
			<td>557226</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD83 APC</td>
			<td>BD Horizon</td>
			<td>551073</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Sep Direct Human Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19669</td>
			<td></td>
		</tr>
		<tr>
			<td>EIF4EBP1 mAb</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>EIF4EBP1 siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-29594</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovin Serum Defined Heat Inactivated</td>
			<td>Hyclone</td>
			<td>SH30070.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>B43618</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH mAb</td>
			<td>Santa Cruz</td>
			<td>SC-47724</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>HuFcR Binding Inhibitor</td>
			<td>eBiosciences</td>
			<td>14-9161-73</td>
			<td>Flow cytometry- blocking buffer</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>B16406</td>
			<td>Software to analyze flow cytometry data</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O55:B5</td>
			<td>Sigma</td>
			<td>L4524</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA microRNA Mimic hsa-miR-146a-5p</td>
			<td>Qiagen</td>
			<td>YM00472124</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION miRNA Negative Control</td>
			<td>Sigma</td>
			<td>HMC0002</td>
			<td>Scrambled miRNA conjugated with a near infrared dye</td>
		</tr>
		<tr>
			<td>Nunc 35mm Cell Culture Dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>R&#38;D Systems</td>
			<td>204-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>R&#38;D Systems</td>
			<td>216-MC-025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-Glutamine</td>
			<td>Corning</td>
			<td>10040CVMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scrambled Control siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-37007</td>
			<td></td>
		</tr>
		<tr>
			<td>Viromer Blue Transfection Reagent Kit</td>
			<td>Lipocalyx</td>
			<td>VB-01LB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1 Cell Proliferation Reagent</td>
			<td>Roche</td>
			<td>5015944001</td>
			<td>Colorimetric assay to assess cell viability</td>
		</tr>
	</tbody>
</table>

isolation, transfection, and culture of primary human monocytes

인간 면역 결핍 바이러스 (HIV)는 1990 년대 중반에 결합 된 항 레트로 바이러스 치료 (cART)의 도입에도 불구하고 주요 건강 관심사로 남아 있습니다. 항레트로바이러스 요법은 전신 바이러스 부하를 효율적으로 낮추고 정상적인 CD4+ T 세포 수를 회복시키지만 완전히 기능적인 면역 체계를 재구성하지는 않습니다. cART를 겪고 있는 HIV 감염한 개별에 있는 역기능 면역 계통은 면역 성 활성화, 면역 세포의 조기 노화, 또는 지속적인 염증에 의해 특징지어질 수 있습니다. 이러한 조건은 HIV 감염과 관련된 comorbid 요인과 함께 세포 및 동물 모델에서 쉽게 재현 할 수없는 질병에 복잡성을 추가합니다. 이 환자에 있는 면역 기능 장애의 근본적인 분자 사건을 조사하기 위하여는, 시험관에서 인간 1 차적인 단핵구를 문화로 조작하는 체계는 여기에서 제시됩니다. 구체적으로, 이 프로토콜은 HIV 음성 대조군뿐만 아니라 CART를 받은 HIV 감염 개인으로부터 얻은 1차 CD14+ 단핵구의 배양 및 형질감염을 허용한다. 이 방법은 단핵구 및 단핵구 유래 대식세포의 격리, 배양 및 형질취득을 포함합니다. 시판되는 키트와 시약이 사용되는 동안, 프로토콜은 miRNA 모방 및 억제제뿐만 아니라 siRNAs와 단핵구의 성공적인 준수 및 형질감염을위한 중요한 팁과 최적화 된 조건을 제공합니다.

설명된 절차를 사용하여, HIV에 감염된 개별 및 건강한 기증자에게서 1 차적인 인간 단핵구는 격리되었습니다. 여기에 제시된 모든 데이터는 낮은(&20 카피/mL) 또는 탐지할 수 없는 바이러스 부하 및 일반 CD4+ T 세포 수로 cART를 받는 HIV+ 피험자로부터 얻어졌습니다. 격리 직후, 세포를 염색하고, 세포 집단의 순도를 확인하기 위해 유세포측정을 수행하였다. ?...

Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Isolation, Transfection, and Culture of Primary Human Monocytes

여기에 제시된 것은 HIV에 감염된 개인과 건강한 대조군으로부터 인간의 1차 단핵구를 분리, 배양, 트랜스펙팅 및 분화하기 위한 최적화된 프로토콜입니다.

1차 인간 단핵구의 고립, 형질전환 및 문화

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

HIV 감염은 검출 가능한 바이러스없이 항 레트로 바이러스 치료를 받는 개인에서도 면역 기능 장애를 일으킵니다. 이 방법은 면역 반응의 주요 조절기인 단세포 기능의 연구를 가능하게 합니다. 우리는 인간의 기본 단핵구의 격리, 문화 및 트랜스퍼팅을 위해 이 기술을 최적화했습니다.우리는 HIV 감염한 개별에 있는 면역 기능 장애의 분자 기계장치를 이해하기 위하여 이 방법을 이용합니다, 그러나 단색세포와 관련되었던 그밖 면역 연구 결과에 적용될 수 있습니다. 이것이 인간의 혈액으로 일하는 첫번째 시간인 경우에, 적당한 생물안전 프로토콜을 따르고 가능하게 전염성 물질로 견본전부를 취급하는 것을 기억하십시오. 4개의 10 밀리리터 EDTA 진공 관에서 40밀리리터의 신선한 인간 전혈을 수집한 후, 멸균 기술을 사용하여 모든 혈액을 생물 안전 캐비닛에 있는 50밀리리터 원추형 프로필렌 튜브로 이송합니다.선택한 인간 단화 절연 키트의 제조업체의 지시에 따라 키트에서 혈액 튜브에 단핵 분리 칵테일 2 밀리리터를 추가하고 키트에서 자기 구슬을 30초 동안 소용돌이시다. 두 밀리리터의 구슬을 혈액에 넣고 25밀리리터 세로지피펫을 사용하여 구슬을 혈액과 조심스럽게 섞습니다. 실온에서 5분 후, 혈액을 50밀리리터 튜브 4개 사이에 균등하게 분할하고 각 튜브에 1밀리미터 EDTA로 보충된 멸균 PBS 30밀리리터를 추가합니다.플라스틱 25 밀리리터 세로지오피펫과 다시 섞어 튜브를 자기 홀더에 놓습니다. 10분 후, 파이펫을 사용하여 각 튜브의 중앙에서 1밀리리터 이상의 적혈구를 흡인하지 않도록 주의하고 튜브 내용물 4개를 새로운 50밀리리터 튜브 중 하나로 분배합니다. 각 튜브에 소용돌이가 있는 자기 구슬의 500마이크로리터를 추가하고 세포 용액을 부드럽게 혼합합니다.튜브를 5분 동안 자기 홀더에 다시 넣은 후 각 튜브의 중앙에서 4개의 새로운 50밀리리터 튜브 중 하나로 조심스럽게 내용물의 내용을 전달합니다. 모든 세포 현탁액이 전달되면 각 새로운 50 밀리리터 튜브를 자석 홀더에 5분 간 배치한 후 튜브 내용내용을 4개의 새로운 50밀리리터 튜브 의 세 번째 세트로 조심스럽게 옮기십시오. 그런 다음 원심 분리에 의해 세포를 수집하고 계산을위한 멸균 PBS의 총 10 밀리리터에서 모든 4 개의 세포 펠릿을 재중단합니다.계산 후, 37섭씨 37도의 혈청 프리 RPMI 1640 배지의 밀리리터 당 10번에서 6번째 세포로 1회 10회에서 분리된 단핵구를 재연하여 항생제로 보충하고 생물안전 성 캐비닛에 있는 35mm 플레이트로 재중단된 세포의 1밀리리터를 플레이트로 플레이트한다. 그런 다음 세포 배양 인큐베이터에 플레이트를 30-60분 동안 배치하여 세포가 우물 바닥에 부착할 수 있도록 합니다. M1과 같은 표현형을 가진 대식세포에 대한 프라이머, 각 접시에 GM-CSF의 밀리리터 당 25 나노그램을 포함하는 태아 소 혈청의 100 마이크로 리터를 추가합니다.M2와 같은 표현형을 가진 대식세포에 대한 프라이머, 각 플레이트에 M-CSF의 밀리리터 당 50 나노그램을 포함하는 태아 소의 100 마이크로 리터를 추가합니다. microRNA 모방, 억제제 또는 작은 간섭 RNA를 가진 단핵 환전의 경우, 트랜스페션 키트에 대한 제조업체의 프로토콜에 따라, 10 마이크로리터의 희석 모방 또는 제5 세포의 10 마이크로리터에서 완충제에서 선택된 RNA를 희석시켰다. 경질 시약을 준비하기 위해 제공된 폴리머의 마이크로리터 1개를 1.5 밀리리터 미세센심분리기 튜브에 추가하고 즉시 키트 제공 버퍼의 90 마이크로리터를 추가하여 트랜지션당 총 91마이크로리터의 시약을 얻습니다.3~5초 동안 리젠트를 소용돌이시키고 희석 된 RNA 10 마이크로리터를 포함하는 튜브내로 전환 용액의 파이펫 90 마이크로리터를 소용돌이시다. 부드러운 혼합 후 실온에서 15분 동안 용액을 배양한 후 100마이크로리터의 트랜스페션 복합체를 하나의 셀에 추가합니다. 섭씨 37도에서 4시간 후, 배지를 적절한 성장 인자를 포함하는 완전한 중간 크기의 3밀리리터로 교체하십시오.문화의 여섯 날에, 태아 소 세럼, 항생제, 리포 폴리사카라이드, 인터페론 감마로 보충 된 매체의 3 밀리리터로 M1 배양 문화 의 초월제를 대체하고 태아 소 혈청, 항생제, M-CSF, 및 인터루크4로 보충 매체로 M2 문화권에서 초퍼를 대체합니다. 24시간 의 배양 후, 활성화된 대식세포 의 각 플레이트를 세척당 신선한 PBS로 두 번 씻으시면 씻습니다. RNA 및 단백질 분석을 위해, 부착된 세포를 플레이트에 직접 리세하여 분석을 위해 방출된 세포 내용물의 수집을 허용한다.유동 세포 측정 분석을 위해 PBS에서 밀리머 EDTA 2개를 10분 동안 섭씨 37도에서 분리한 후 플레이트 바닥에서 세포를 부드럽게 긁어 내어 수집을 1.5 밀리리터 미세원심분리기로 분석합니다. 여기서 는 비활성화 T0 세포에 비해 CD163 및 CD209의 증가된 수준을 가진 CD80 및 CD83 및 M2 활성화 세포의 증가된 수준을 가진 M1 활성화 제어 및 환자 유래 세포의 대표적인 히스토그램이 도시된다. 흥미롭게도, CD80 및 CD83은 HIV 유래 세포에 비해 대조군 유래 세포에서 더 높게 발현되는 것으로 나타났다.스크램블근적외선 표지 된 microRNA를 사용하여 갓 수집 된 단핵구의 트랜스페션은 유동 세포 측정 및 공초점 현미경 검사법에 의해 결정된 바와 같이 90 % 이상의 트랜스페션 효율을 초래합니다. 또한, 형질전환은 세포 성숙 또는 경질 조건의 단계에 관계없이 환자 유래 또는 제어 세포의 생존가능성을 크게 감소시키지 않는다. 트랜스펙트 결과 1일 및 4일째 사후 격리에서 특정 소형 간섭 RNA를 통해 트랜스포팅 시 표적 유전자의 효과적인 다운레충이 초래됩니다.또한, microRNA 모방으로 감염된 세포는 모든 트랜스퍼션 컨트롤이 유의할 수 없는 변화를 보이지 않는 동안, 미감염 된 세포에 대한 microRNA 발현의 48 ~ 72 배 증가를 입증한다. 도금된 세포의 수는 생존에 매우 중요합니다. 35mm 접시당 100만 개의 셀을 권장합니다.혈청이 없는 배지로 도금도 세포 부착을 크게 향상시킬 것입니다. 이 방법으로 얻은 세포는 관심있는 환자와 건강한 통제의 환자에 있는 차동 반응을 공부하기 위하여 사이토카인 또는 성장 인자로 취급될 수 있습니다.

isolation-transfection-and-culture-of-primary-human-monocytes

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

마이 그 레이션의 인간 T 림프구 분리, 문화 및 분석 체외에서</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

연구

면역학 및 감염병학

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 ...

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 ...

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. ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

표현형 분석 및 대한 인간의 피부에서 림프구 분리<em&gt; 전의 VIVO</em&gt; 세포 배양

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

에 대한 응답으로 인간의 기본 호중구와 단핵구에 의해 항진균 활성의 라이브 영상<em&gt;를했다. 투스</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

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

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

중생의 연구에 대 한 기본 위 상피 단층 문화 마우스 및 인간의 파생

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...