Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method

Abstract

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo®). Here we describe a public domain "Dye Drop" method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.

Competing Interest Statement

PKS is a co-founder and member of the BOD of Glencoe Software, a member of the BOD for Applied Biomath, and a member of the SAB for RareCyte, NanoString and Montai Health; he holds equity in Glencoe, Applied Biomath and RareCyte. PKS is a consultant for Merck and the Sorger lab has received research funding from Novartis and Merck in the past five years. Sorger declares that none of these relationships have influenced the content of this manuscript. KS is currently an employee of Bristol Myers Squibb, MH and LG are currently employees of Genentech, and MN is currently an employee of Ribon Therapeutics. The other authors declare no outside interests.

Footnotes

* Panels were added to Figures 1, 3 and 5. Supplementary Figure 2 was added. Additional benchmarking of the method was added, as was a detailed breakdown of assay costs in Supplementary Table 1.

* https://labsyspharm.shinyapps.io/HMSLINCS_BRCA_Browser/

* https://www.synapse.org/#!Synapse:syn26133007