An Optimization Method of PBMC Staining and Visualization Using Fluorescence Microscopy

Fluorescence microscopy (FM) utilizes fluorescent staining to label intra- and extracellular molecules, offering high-resolution imaging capabilities and facilitating the study of various pathological processes. This study aims to optimize staining and imaging protocols for peripheral blood mononuclear cells (PBMC) using FM, which is crucial for obtaining reliable and reproducible results. After optimization, FM could become one of the fundamental research techniques focused on understanding immune cell function. Despite extensive research, there is no standard PBMC staining protocol for FM, which still generates many issues with this technique's fast and proper usage. We aim to bridge this gap through systematic optimization of each step of the staining and imaging process. Our research includes selecting the most suitable fluorescent dyes, optimizing fixation and permeabilization protocols, and studying imaging parameters. The aim is to improve the signal-to-noise ratio and obtain high-resolution images of stained PBMC. By developing a standard methodology, this research may contribute to the advancement of FM-based techniques for PBMC, offering researchers more possibilities for exploring the immune system.