Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies. Issue 7 (12th February 2020)

• Record Type: Journal Article
• Authors: Smiddy, Nicole M.; DiSalvo, Matthew; Allbritton-King, Jules D.; Allbritton, Nancy L.
• Is Part Of: Analyst. Volume 145:Issue 7(2020)
• Page Start: 2649
• Page End: 2660
• Publication Date: 2020-02-12
• Subjects: Chemistry, Analytic -- Periodicals; 543
• DOI: 10.1039/d0an00030b
• Languages: English
• ISSNs: 0003-2654

Abstract

Abstract : A magnetic, microwell-based plate was established enabling automated collection of colony biopsy fragments into known locations for immunofluorescence assays. Abstract : 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.