Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi‐pass acquisition

Abstract The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins (FPs), and can destroy chemically‐sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi‐pass flow cytometry approach to address this long‐standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single‐cell resolution maintained. Chemically‐fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular FPs and methanol‐sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cellular analysis without the constraints of conventional one‐time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno‐oncology, stem cell research, and cell biology.