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<p>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.</p>

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Comprehensive analysis of cellular metrics: From proliferation to mitochondrial membrane potential and cell death in a single sample

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年3月24日

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  • Sirina Sabirova,
  • Gulnaz Sharapova,
  • Aida Budyukova,
  • Marina Gomzikova,
  • Anna Brichkina,
  • Nick A. Barlev,
  • Albert Rizvanov,
  • Nikita Markov,
  • Hans-Uwe Simon

摘要

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.