Standardized Workflow for High-Content Imaging and Data Processing.

To address the challenges of low reproducibility due to operational variations in scientific research imaging and the complexity of analyzing a large number of samples using conventional methods, we introduce a protocol for staining HT22 cells with PI and Hoechst 33342 and then imaging and analyzing the data using the High-Content Imaging System. This workflow aims to establish robust imaging setting parameters and stringent quality control standards. It demonstrates the instrument's imaging settings and the workflow for three different image processing methods provided by the instrument: Multi-Wavelength Cell Scoring (Multi), Live-Dead (Live), and Custom Module Editor (CME). The image processing methods are compared and validated against the traditional method, ImageJ, to ensure their effectiveness. The study ultimately summarizes a workflow for using the High-Content Imaging System to determine cell viability using the PI and Hoechst double-staining method. By standardizing the entire imaging and data processing workflow, the study significantly enhances the reproducibility and reliability of scientific research imaging, thereby providing solid methodological support for cross-laboratory data comparison and advancing scientific research.