Optimisation of a screening platform for determining IL-6 inflammatory signalling in the senescence-associated secretory phenotype (SASP)
Optimisation of a screening platform for determining IL-6 inflammatory signalling in the senescence-associated secretory phenotype (SASP)
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DOI：
10.1007/s10522-019-09796-4
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发表时间：
2019-02
期刊：
影响因子：
4.5
通讯作者：
A. Rolt;A. Nair;L. Cox
中科院分区：
文献类型：
--
作者：
A. Rolt;A. Nair;L. Cox
细胞衰老已被证明足以发展多种年龄相关的病理。衰老细胞采用分泌表型（SASP），其包含大量促炎细胞因子、趋化因子和蛋白酶。SASP本身被认为是年龄相关疾病的许多病理学的病因，并且对开发可以抑制SASP的衰老修饰剂的兴趣越来越大。然而，为了鉴定新的药剂，有必要使用稳健的测定法进行中到高通量筛选以获得所需的结果。在这里，我们描述了基于细胞的生物传感器HEK细胞系的优化和验证，用于测量由原代衰老成纤维细胞分泌到条件培养基中的范围内的IL-6浓度，适用于适合于文库筛选应用的384孔板格式。我们进一步表明，该测定可以测量依赖于细胞群体年龄的IL-6分泌的变化，并且该测定响应于衰老细胞中的mTOR抑制，这降低了SASP，包括IL-6。因此，我们建议，这种优化的生物传感器，我们称之为HEK-SASP，可能会证明在研究中需要强大的，可再生的和相对便宜的测定SASP因素的价值。
Cellular senescence has been shown to be sufficient for the development of multiple age-related pathologies. Senescent cells adopt a secretory phenotype (the SASP) which comprises a large number of pro-inflammatory cytokines, chemokines and proteases. The SASP itself is thought to be causative in many pathologies of age-related diseases, and there is growing interest in developing seno-modifying agents that can suppress the SASP. However, in order to identify new agents, it is necessary to conduct moderate to high throughput screening with robust assays for the required outcome. Here, we describe optimisation and validation of a cell-based biosensor HEK cell line for measurement of IL-6 concentrations within the range secreted into conditioned medium by primary senescent fibroblasts, adapted for a 384 well plate format suitable for library screening applications. We further show that the assay can measure changes in IL-6 secretion dependent on cell population age, and that the assay is responsive to mTOR inhibition in the senescent cells, which reduces the SASP, including IL-6. Hence, we propose that this optimised biosensor, which we term HEK-SASP, may prove of value in studies requiring robust, renewable and relatively inexpensive assays for measuring SASP factors.