Development of a Plate-Based Assay for High-Content Analysis of Individual Untethered Non-Adherent Cells

Research involving non-adherent cell lines, primary cells and blood cells is definitely important, but its application in image-based assays, especially in high-content systems, is highly limited. Accordingly, efficient highcontent methods to study non-adherent cells are needed not only to improve diagnostics but also for early screening of targeted drugs. A plate-based assay using adhesion reagents for multiparametric measurement with single non-adherent and non-anchored cells in a large cell population in high-content cytometry was developed and optimized. The cells preserved their identity even during extensive biomanipulations. The proposed method is highly robust for better imaging and can be used in various assays in different cellular backgrounds. Furthermore, as exemplary experiments, novel optimized assay protocols were used to study extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity after cell inhibition with imatinib in chronic myelocytic leukemia K562 cells, revealing the phosphorylation kinetics of ERK MAPK. The results showed that the proposed assay detects kinase phosphorylation with good sensitivity and may be used in rapid drug screening. Keywords: Development and optimization, high-content analysis, non-adherent cells, adhesion reagents, cell fixation, primary cells, image-based assays, extracellular signal-regulated kinase, drug screening, leukemia, phosphorylation, adhesive protein, Cell Viability