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eFluor 450 Enables Superior Detection of Intracellular Staphylococcus aureus in High-Autofluorescence Triple-Negative
Sima Kianpour Rad1,2, Runhao Li1,2, Kenny K L Yeo1,2
1Solid Tumour Group, Basil Hetzel Institute for Translational Health Research, The Queen Elizabeth Hospital, Central Adelaide Local Health Network, Adelaide, South Australia, Australia.
Summary
eFluor 450 dye offers superior bacterial detection in cancer cells compared to CFSE, improving accuracy in flow cytometry analyses. This fixable dye enhances discrimination of intracellular bacteria, crucial for studying microbial invasiveness.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Distinguishing extracellular from intracellular bacteria is vital for assessing microbial invasiveness.
- Flow cytometry is a powerful tool for high-throughput analysis but faces challenges with high autofluorescence in tumor cells.
- Accurate bacterial detection is essential for understanding host-pathogen interactions in cancer.
Purpose of the Study:
- To compare the efficacy of eFluor 450 and carboxyfluorescein succinimidyl ester (CFSE) for labeling Staphylococcus aureus during intracellular infection of triple-negative breast cancer cells.
- To evaluate dye performance in spectral flow cytometry, particularly in the context of high cellular autofluorescence.
- To identify an improved fluorescent dye for precise bacterial detection in cancer models.
Main Methods:
- Intracellular labeling of Staphylococcus aureus in triple-negative breast cancer cell lines using eFluor 450 and CFSE.
- Flow cytometry analysis with spectral unmixing to quantify bacterial load and assess population separation.
- Comparison of dye performance based on fluorescence intensity, population discrimination, labeling time, reagent volume, and bacterial viability.
Main Results:
- eFluor 450 demonstrated superior population separation and higher estimated fluorochrome abundance compared to CFSE, even in highly autofluorescent cells.
- The improved performance of eFluor 450 is attributed to better spectral separation from cellular autofluorescence.
- eFluor 450 required less labeling time and reagent volume than CFSE without affecting bacterial viability, with benefits most evident at low bacterial loads.
Conclusions:
- eFluor 450 is an effective alternative to CFSE for detecting intracellular bacteria in cancer models using spectral flow cytometry.
- The spectral properties of fluorochromes are critical for accurate host-pathogen analyses, especially in complex biological samples.
- This study establishes eFluor 450 as a valuable tool for precise microbial invasiveness assessment in cancer research.
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