Evaluating cell viability assessment techniques: a comparative study of flow cytometry and fluorescence microscopy in response to bioactive glass exposure

Reliable in vitro cytotoxicity assessment of biomaterials is essential for preclinical evaluation. Fluorescence microscopy (FM) and flow cytometry (FCM) are widely used methods, yet their comparative performance in particulate systems remains underexplored. This study compares FM and FCM in evaluating the cytotoxicity of Bioglass 45S5 (BG) on SAOS-2 osteoblast-like cells across different particle sizes and concentrations. The cells were treated with BG particles in three size ranges (< 38 µm, 63-125 µm, and 315-500 µm) at concentrations of 25, 50, and 100 mg/mL for 3 and 72 h. FM employs FDA/PI staining to distinguish viable and nonviable cells, whereas FCM utilizes multiparametric staining (Hoechst, DiIC1, Annexin V-FITC, and PI) to classify viable, apoptotic, and necrotic populations. Both techniques confirmed a clear trend: smaller particles and higher concentrations caused greater cytotoxicity. The most pronounced effect was observed for < 38 µm particles at 100 mg/mL, which reduced FM-assessed viability to 9% at 3 h and 10% at 72 h; FCM measurements under the same conditions revealed 0.2% and 0.7% viability, respectively. The controls retained > 97% viability, indicating size-based cytotoxicity. A strong correlation between FM and FCM data (r = 0.94, R² = 0.8879, p < 0.0001) was observed. FCM further distinguished early and late apoptosis from necrosis and demonstrated superior precision, particularly under high cytotoxic stress. These findings highlight the size- and dose-dependent cytotoxic potential of BG and support the use of FCM as a robust, quantitative tool for cytocompatibility evaluation in particulate biomaterial research.