Introduction and objectives:

Over the past two decades, flow cytometry has undergone major technological advances, including miniaturization and the development of high-throughput screening (HTS) capabilities [1, 2]. These improvements have increased accessibility, sensitivity, and multiparametric analysis potential. As a result, flow cytometry is now widely used in fields ranging from eukaryotic cell biology to microbiology, pharmacology, and environmental sciences.

Since 2007, the eBioCyt UPS1401 platform (University of Strasbourg) has developed miniaturized fluorescence-based assays for both eukaryotic and prokaryotic cells [3–5]. This work highlights the role of flow cytometry in interdisciplinary projects aimed at developing tools for cellular and bacterial assays, including probe–bacteria interactions, antibacterial activity of natural compounds, cytotoxicity of pollutants, and oxidative stress in mammalian cells.

Materials and Methods:

Gram-positive and Gram-negative bacteria were incubated with fluorescent probes, including Nile Red-based antimicrobial peptides and the solvatochromic peptide UNR-1, enabling wash-free staining analyzed by flow cytometry. Antibacterial activity of natural compounds, such as sesquiterpene coumarins from Ferula communis L., was evaluated using dose–response experiments and propidium iodide staining to assess viability. For environmental applications, pollutant cytotoxicity, including asbestos-containing waste, was assessed on mammalian cell lines. Oxidative stress assays were performed to quantify reactive oxygen species (ROS) production and evaluate cytotoxicity after exposure to synthetic molecules.

Results and discussion:

Flow cytometry enabled rapid and sensitive characterization of cellular responses across all projects. Fluorescent probes showed differential bacterial labeling, with UNR-1 (bearing Alared) exhibiting the highest efficiency [6, 7]. Dose–response relationships were established for several bacteriotoxic compounds, and sesquiterpene coumarins demonstrated antibacterial activity [8].

In mammalian cell lines, flow cytometry allowed quantification of oxidative stress and cytotoxicity, supporting the development of HTS-compatible assays for drug evaluation. In environmental applications, a bioremediation strategy showed promising efficiency in removing iron from asbestos-containing waste.

Conclusion:
Overall, this work demonstrates the versatility of flow cytometry as a central tool for high throughput applied research in pharmacology and environmental sciences. Future work will focus on validating the bioremediation approach and assessing its impact on cytotoxicity reduction.

1 Peluso J, Muller CD. Advances in flow cytometry for drug screening. Expert Opin Drug Discov. 2010 Sep;5(9):827-33. doi: 10.1517/17460441.2010.509395.

2 Peluso J, Tabaka-Moreira H, Taquet N, Dumont S, Muller CD, Reimund JM. Can flow cytometry play a part in cell based high-content screening? Cytometry A. 2007 Nov;71(11):901-4. doi: 10.1002/cyto.a.20455.

3 Hulot C, Peluso J, Blond G, Muller CD, Suffert J. Synthesis of exotic polycycles such as cyclooctatrienes and fenestrenes with differential pro-apoptotic activities on human TRAIL-resistant metastatic cell lines. Bioorg Med Chem Lett. 2010 Nov 15;20(22):6836-9. doi: 10.1016/j.bmcl.2010.08.094.

4 Molnár F, Sigüeiro R, Sato Y, Araujo C, Schuster I, Antony P, Peluso J, Muller C, Mouriño A, Moras D, Rochel N. 1α,25(OH)2-3-epi-vitamin D3, a natural physiological metabolite of vitamin D3: its synthesis, biological activity and crystal structure with its receptor. PLoS One. 2011 Mar 31;6(3):e18124. doi: 10.1371/journal.pone.0018124.

5 Carré G, Benhamida D, Peluso J, Muller CD, Lett MC, Gies JP, Keller V, Keller N, André P. On the use of capillary cytometry for assessing the bactericidal effect of TiO2. Identification and involvement of reactive oxygen species. Photochem Photobiol Sci. 2013 Apr;12(4):610-20. doi: 10.1039/c2pp25189b.

6 Weiss L, Mirloup A, Blondé L, Manko H, Peluso J, Bonnet D, Dziuba D, Karpenko J. Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-Free Staining of Bacteria. Bioconjug Chem. 2024 Nov 20;35(11):1779-1787. doi: 10.1021/acs.bioconjchem.4c00331.

7 Weiss L, Bonnet D, Dziuba D, Karpenko J. Flow Cytometry Analysis of Perturbations in the Bacterial Cell Envelope Enabled by Monitoring Generalized Polarization of the Solvatochromic Peptide UNR-1. Anal Chem. 2025 Jan 14;97(1):622-628. doi: 10.1021/acs.analchem.4c04953.

8 Braillon C, Aubert E, Janet-Bintz R, Gjikolaj M, Hamidi R, Blondé L, Peluso J, Villa P, Pitchon V, Fechter P, Vonthron-Sénécheau C, Ortiz S. Targeted isolation of new antibacterial sesquiterpene coumarins from ammoniacum (Ferula communis L.). Fitoterapia. 2025 Oct;186:106833. doi: 10.1016/j.fitote.2025.106833. Epub2025 Aug 17.