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首页> 外文期刊>Immunology: An Official Journal of the British Society for Immunology >Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro in vitro
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Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro in vitro

机译：单核细胞隔离技术显着影响分离的单核细胞的表型，体外体外衍生巨噬细胞

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Summary Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14 pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14 pos selection resulted in highest monocyte yield (19·8?×?10 6 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7?×?10 6 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1?×?10 6 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14 pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14 pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80 high ?HLA‐DR high ?CD163 low ), whereas negative selection MDMs were M2‐skewed (CD80 low ?HLA‐DR low ?CD163 high ). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.
机译：发明内容从外周血单核细胞产生的单核细胞衍生的巨噬细胞（MDMS）被广泛用于模拟人类巨噬细胞的体外研究。然而，不同隔离方法对所得MDM表型的可能影响很差。我们的目标是探讨三种常用的单核细胞分离技术对所得MDM表型的影响。比较塑料粘附，阴性选择和CD14 POS选择。用巨噬细胞和粒细胞 - 巨噬细胞殖民地刺激因子的5天培养产生单核细胞衍生的巨噬细胞。我们研究了单核细胞和MDM产量，纯度，活力和细胞表型。 CD14 POS选择导致最高单核细胞产率（19·8？×10 6个细胞，相当于总量的70％）和纯度（98·7％），与阴性选择相比（17·7？×10 6个细胞，总总量的61％，纯度为85·0％）和塑料粘附（6·1？×10 6个细胞，12·9％总量，44·2％纯度）。选择性选择的单核细胞受到血小板的高度污染。与未触及的外周血单核细胞相比，CD14 POS选择和塑料粘合性单核细胞的表达显着降低。成熟后，CD14 POS选择也导致最高的MDM纯度（98·2％）与阴性选择（94·5％）和塑料粘附（66·1％）。此外，来自塑料粘附的MDMS是M1倾斜的（CD80高？HLA-DR高？CD163低），而负选择MDMS为M2偏斜（CD80低ΔHLA-DR低温）。单核细胞隔离方法的选择不仅显着影响产量和纯度，而且影响培养的MDMS的结果表型。可以部分地通过在使用塑料粘附或阴性选择时存在污染细胞来分散地解释这些差异。仔细考虑单核细胞隔离方法对于在MDMS上设计体外测定是重要的。

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