eFluor 450 Enables Superior Detection of Intracellular Staphylococcus aureus in High‐Autofluorescence Triple‐Negative Breast Cancer Cells

Accurate discrimination of extracellular from intracellular bacteria is critical for assessing microbial invasiveness in host cells. Flow cytometry offers high-throughput, quantitative analysis but can be confounded by high autofluorescence in certain tumor-derived cell types. This study compared the performance of two fluorescent dyes, carboxyfluorescein succinimidyl ester (CFSE), a widely used cell-permeant dye, and eFluor 450, a fixable amine-reactive dye, for labeling Staphylococcus aureus during intracellular infection of triple-negative breast cancer cell lines. Both dyes produced multiplicity-of-infection-dependent increases in fluorescence; however, eFluor 450 consistently achieved superior population separation and higher estimated fluorochrome abundance following spectral unmixing, even in highly autofluorescent backgrounds. These differences reflect more favorable spectral separation from cellular autofluorescence rather than generic signal enhancement. Operationally, eFluor 450 required shorter labeling time and lower reagent volume than CFSE without compromising bacterial viability. The advantages of eFluor 450 were most pronounced at low bacterial loads and in applications requiring precise population discrimination. To our knowledge, this is the first demonstration of eFluor 450 for intracellular bacterial detection by spectral flow cytometry in cancer models. Together, these findings establish eFluor 450 as an efficient alternative to conventional dyes and highlight the importance of fluorochrome spectral properties for unmixing-based host-pathogen analyses.