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<p>Flow cytometry is a critical technology for single-cell analysis; however, sorting large particles (&gt; 30&#xa0;μm) remains challenging owing to low recovery rates. This study investigated the relationship between particle size and the Break-off point (BOP) and assessed the impact of large particles on droplet formation through a combination of simulations and experiments. The results revealed that interference between the particle-induced BOP and vibration-driven BOP leads to droplet instability—identified as the key factor limiting recovery. We introduce ΔBOP as the difference between these two BOPs and propose an optimization strategy to stabilize droplet formation. Implementing this ΔBOP-based approach increased recovery from 72.2% (794/1100) to 92.4% (1016/1100) for 35-µm particles and from 23.0% (115/500) to 75.6% (378/500) for 50-µm particles. This strategy demonstrates broad applicability for high-throughput single-cell functional analyses involving various particle types, including hydrogel particles, double emulsions, and agarose beads, and opens new possibilities for applications such as secretion profiling and intercellular communication research.</p>

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Enhancing large particle recovery in high-throughput functional cell sorting through ΔBOP optimization

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年12月21日

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  • Naohisa Sakamoto,
  • Eikichi Shibata,
  • Mitsuo Yoshimura,
  • Haruto Namura,
  • Yudai Yanashita,
  • Shin Masuhara,
  • Yusuke Uehara,
  • Kenji Yamane,
  • Hiroto Kasai,
  • Motohiro Furuki,
  • Yoshiharu Hayashi

摘要

Flow cytometry is a critical technology for single-cell analysis; however, sorting large particles (> 30 μm) remains challenging owing to low recovery rates. This study investigated the relationship between particle size and the Break-off point (BOP) and assessed the impact of large particles on droplet formation through a combination of simulations and experiments. The results revealed that interference between the particle-induced BOP and vibration-driven BOP leads to droplet instability—identified as the key factor limiting recovery. We introduce ΔBOP as the difference between these two BOPs and propose an optimization strategy to stabilize droplet formation. Implementing this ΔBOP-based approach increased recovery from 72.2% (794/1100) to 92.4% (1016/1100) for 35-µm particles and from 23.0% (115/500) to 75.6% (378/500) for 50-µm particles. This strategy demonstrates broad applicability for high-throughput single-cell functional analyses involving various particle types, including hydrogel particles, double emulsions, and agarose beads, and opens new possibilities for applications such as secretion profiling and intercellular communication research.