poster discovery
High-content analysis of DNA damage markers in multiplexed cell lines using the SemaCyte microcarrier platform
Jackline Agwenge, Simon Stockwell o2h Discovery, Hauxton House, Mill SciTech Park, Cambridge CB22 5HX
Abstract High-content imaging (HCI) offers a considerable depth of analysis options when considering the requirements for developing a new cellular assay. The information-rich image data collected during an HCI assay allows access to measures of spatial, quantitative, temporal and morphological markers of target engagement, often requiring monolayers of adherent cells seeded to assay plates. The SemaCyte microcarrier system developed by Semarion enables powerful new strategies to manage cell monolayers in drug discovery workflows. In collaboration with Semarion, o2h Discovery have updated an established HCI approach to DNA damage to explore
SemaCyte-enabled enhancements in cellular assays; miniaturisation of seeding, assay-ready adherent cells, multiplexed cell lines and subpopulation barcoding are investigated here. Objectives Perform a first-pass test using the SemaCyte system to obtain and analyse cellular HCI data, making use of cell multiplexing capabilities and same-day set up of assays from frozen cell SemaCyte stocks
Assay details o 96-well plate, 2hr, 11-point etoposide (EP) DRC o Cells presented as pooled SemaCytes in each well o Using frozen SemaCytes on same day as recovery o Fixed endpoint - probes for DNA damage markers o 3 multiplexed cell lines per well – U2OS, A549, U87MG o 4 SemaCyte barcodes used for deconvolution by HCI th o Plate reference control: U2OS cell line re-used for 4 barcode providing fixed 50μM set, spiked to every well
Early analyses with this data set yield sufficient information to characterise and differentiate variability in DNA damage dose responses between cell lines grown in parallel using SemaCytes
Thawed monolayers recovery of frozen assay-ready monolayers worked well (>= 75% viable) and offers a new flexibility to scheduling deployment to assays and maintaining cells for Data handling
The ability to deconvolute mixed populations using image processing to sort SemaCytes by barcodes proved accessible and opens further options for how to organize and control HCI assays
Next steps – A deeper dive into the current data set is planned, including exploration of RAD51 and implementation of dynamic image acquisition strategies such as the CellInsight EurekaScan
3 cell lines: o U2OS o U87MG o A549 Each assigned SemaCyte barcodes Revived following frozen storage Pooled for assay Drugged 2hr with EP DRC same day Fixed and stained for subsequent HCI
2. High-content imaging
Hoeschst 33342 αα RAD51 (AF488)RAD51 (AF488)
10x objective 25 fields per channel Image stitching α γH2AX (AF647)
Phalloidin (iFluor555)
Barcodes (BF)
3. Image processing I – deconvolution QC and extraction of SemaCytes according to cell line-specific barcodes
4. Image processing II – quantitation Cell Profiler to detect and characterise cells within SemaCyte-defined image masks
Cell by cell feature extraction
Acknowledgements o2h Discovery would like to acknowledge the team at Semarion for their collaborative support and insight. Furthermore, ThermoFisher Scientific have provided indispensable support across instrument and data-handling steps in the workflow.
First-pass analysis focused on γH2AX marker of DNA damage Data resolves rank order cell line sensitivities to EP Separately barcoded technical control U2OS 50μM EP population correctly intersect main DRC