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Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies.
Analyst ( IF 3.6 ) Pub Date : 2020/02/06 , DOI: 10.1039/d0an00030b Nicole M Smiddy 1 , Matthew DiSalvo , Jules D Allbritton-King , Nancy L Allbritton
Analyst ( IF 3.6 ) Pub Date : 2020/02/06 , DOI: 10.1039/d0an00030b Nicole M Smiddy 1 , Matthew DiSalvo , Jules D Allbritton-King , Nancy L Allbritton
Affiliation
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The majority of bioassays are cell-lethal and thus cannot be used for cell assay and selection prior to live-cell sorting. A quad microraft array-based platform was developed to perform semi-automated cell sampling, bioassay, and banking on ultra-small sample sizes. The system biopsies and collects colony fragments, quantifies intracellular protein levels via immunostaining, and then retrieves the living mother colonies based on the fragments' immunoassay outcome. To accomplish this, a magnetic, microwell-based plate was developed to mate directly above the microraft array and capture colony fragments with a one-to-one spatial correspondence to their mother colonies. Using the Signal Transducer and Activator of Transcription 3 (STAT3) model pathway in basophilic leukemia cells, the system was used to sort cells based on the amount of intracellular STAT3 protein phosphorylation (pSTAT3). Colonies were detected on quad arrays using bright field microscopy with 96 ± 20% accuracy (true-positive rate), 49 ± 3% of the colonies were identified as originating from a single cell, and the majority (95 ± 3%) of biopsied clonal fragments were successfully collected into the microwell plate for immunostaining. After assay, biopsied fragments were matched back to their mother colonies and mother colonies with fragments possessing the greatest and least pSTAT3/STAT3 were resampled for expansion and downstream biological assays for pSTAT3/STAT3 and immune granule exocytosis. This approach has the potential to enable colony screening and sorting based on assays not compatible with cell viability, greatly expanding the cell selection criteria available to identify cells with unique phenotypes for subsequent biomedical research.
中文翻译：
基于微筏阵列的平台，用于基于微集落活检中细胞内蛋白质的细胞致死免疫分析对可行的微集落进行分选。
大多数生物测定是细胞致死的，因此不能用于活细胞分选之前的细胞测定和选择。开发了基于四微筏阵列的平台，用于执行半自动细胞采样、生物测定和超小样本量银行业务。该系统进行活组织检查并收集集落片段，通过免疫染色定量细胞内蛋白质水平，然后根据片段的免疫测定结果检索活的母集落。为了实现这一目标，开发了一种基于微孔的磁性板，用于直接在微筏阵列上方交配，并捕获与其母菌落具有一对一空间对应关系的菌落片段。该系统利用嗜碱性白血病细胞中的信号转导器和转录激活剂 3 (STAT3) 模型途径，根据细胞内 STAT3 蛋白磷酸化 (pSTAT3) 的量对细胞进行分类。使用明视野显微镜在四元阵列上检测集落，准确度为 96 ± 20%（真阳性率），49 ± 3% 的集落被鉴定为源自单个细胞，并且大多数（95 ± 3%）的活检克隆片段成功收集到微孔板中进行免疫染色。测定后，将活检片段与其母集落匹配，并对具有最大和最小 pSTAT3/STAT3 的片段的母集落重新取样，用于 pSTAT3/STAT3 和免疫颗粒胞吐作用的扩展和下游生物测定。这种方法有可能实现基于与细胞活力不兼容的测定的集落筛选和分选，从而极大地扩展了可用于识别具有独特表型的细胞以用于后续生物医学研究的细胞选择标准。
更新日期：2020-03-31
中文翻译：
基于微筏阵列的平台，用于基于微集落活检中细胞内蛋白质的细胞致死免疫分析对可行的微集落进行分选。
大多数生物测定是细胞致死的，因此不能用于活细胞分选之前的细胞测定和选择。开发了基于四微筏阵列的平台，用于执行半自动细胞采样、生物测定和超小样本量银行业务。该系统进行活组织检查并收集集落片段，通过免疫染色定量细胞内蛋白质水平，然后根据片段的免疫测定结果检索活的母集落。为了实现这一目标，开发了一种基于微孔的磁性板，用于直接在微筏阵列上方交配，并捕获与其母菌落具有一对一空间对应关系的菌落片段。该系统利用嗜碱性白血病细胞中的信号转导器和转录激活剂 3 (STAT3) 模型途径，根据细胞内 STAT3 蛋白磷酸化 (pSTAT3) 的量对细胞进行分类。使用明视野显微镜在四元阵列上检测集落，准确度为 96 ± 20%（真阳性率），49 ± 3% 的集落被鉴定为源自单个细胞，并且大多数（95 ± 3%）的活检克隆片段成功收集到微孔板中进行免疫染色。测定后，将活检片段与其母集落匹配，并对具有最大和最小 pSTAT3/STAT3 的片段的母集落重新取样，用于 pSTAT3/STAT3 和免疫颗粒胞吐作用的扩展和下游生物测定。这种方法有可能实现基于与细胞活力不兼容的测定的集落筛选和分选，从而极大地扩展了可用于识别具有独特表型的细胞以用于后续生物医学研究的细胞选择标准。
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