Isolation, Transfection, and Culture of Primary Human Monocytes.
Isolation, Transfection, and Culture of Primary Human Monocytes.
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原代人单核细胞的分离，转染和培养。
DOI：
10.3791/59967
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发表时间：
2019-12-16
期刊：
影响因子：
--
通讯作者：
Peruzzi F
中科院分区：
文献类型：
--
作者：
Plaisance-Bonstaff K;Faia C;Wyczechowska D;Jeansonne D;Vittori C;Peruzzi F
Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.
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