Comprehensive analysis of cellular metrics: From proliferation to mitochondrial membrane potential and cell death in a single sample
Sabirova S. Sharapova G. Budyukova A. Gomzikova M. Brichkina A. Barlev N.A. Rizvanov A. Markov N. Simon H.-U.
December 2025Springer Nature
Cell Death Discovery
2025#11Issue 1
Changes in cell number during in vitro experiments and pharmacological screenings primarily depend on two factors: cell death and proliferation. The dynamics of these processes determine whether cell populations expand and accumulate or, conversely, decrease over time. Understanding the biological mechanisms governing these changes is crucial for deciphering the mode of action of any pharmacological or genetic treatment in fundamental research and pre-clinical trials. In this context, we introduce a robust and efficient flow cytometry-based methodology that enables comprehensive analysis of key cellular parameters that indicate changes in cell numbers. This approach encompasses the assessment of cell count along with critical maintenance parameters including proliferation, cell cycle dynamics, apoptosis, cell permeability, and mitochondrial depolarization. These parameters are intricately linked, offering a detailed view of the cellular state. The described methodology is versatile and adaptable for analyzing various cell types, whether at steady state or in response to treatments. To develop this workflow, we integrated and optimised multiple flow cytometry-based stainings such as annexin V, propidium iodide, bromodeoxyuridine, CellTrace Violet, and JC-1 into a unified protocol. This article offers a detailed, step-by-step guide to the entire method, covering aspects such as timing, sample preparation techniques, and the reagents used. Additionally, it includes examples of the data that can be obtained with this technique and illustrates its multiparametric visualization. Collectively, this methodology facilitates the rapid acquisition of up to eight different parameters from a single sample in one experiment.
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Laboratory of Molecular Immunology, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation
Laboratory of Intercellular Communication, Kazan Federal University, Kazan, Russian Federation
OpenLab Gene and Cell Technology, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation
Institute of Systems Immunology, Center for Tumor Biology and Immunology, Philipps University of Marburg, Marburg, Germany
Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russian Federation
Department of Biomedical Sciences, School of Medicine, Nazarbayev University, Astana, Kazakhstan
Division of Medical and Biological Sciences, Tatarstan Academy of Sciences, Kazan, Russian Federation
Institute of Pharmacology, University of Bern, Bern, Switzerland
Institute of Biochemistry, Brandenburg Medical School, Neuruppin, Germany
Laboratory of Molecular Immunology
Laboratory of Intercellular Communication
OpenLab Gene and Cell Technology
Institute of Systems Immunology
Institute of Cytology of the Russian Academy of Sciences
Department of Biomedical Sciences
Division of Medical and Biological Sciences
Institute of Pharmacology
Institute of Biochemistry
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