Two-step protocol for preparing adherent cells for high-throughput flow cytometry.

Area of Science:

• Cell Biology
• Biotechnology
• Analytical Chemistry

Background:

• Standard protocols for preparing adherent cells for flow cytometry are often multistep, leading to significant cell loss.
• High-throughput analysis requires efficient and reproducible cell preparation methods.

Purpose of the Study:

• To develop a simple, cost-effective, and labor-efficient two-step protocol for adherent cell preparation for high-throughput flow cytometry.
• To reduce cell loss and improve efficiency compared to standard protocols.

Main Methods:

• Adherent cells were cultured in microplates and detached using EDTA directly in cell culture medium.
• Cells were stained and analyzed using flow cytometry, bypassing washing, centrifugation, and plate transfers.

Main Results:

• The two-step protocol was validated across six adherent cell lines, four dyes, and two antibodies.
• The method demonstrated reduced cell loss and was confirmed on two different flow cytometry instruments.

Conclusions:

• This optimized protocol provides a simplified and efficient approach for adherent cell preparation in high-throughput flow cytometry.
• The method is suitable for analyzing various cellular functions, including apoptosis, mitochondrial membrane potential, reactive oxygen species, and autophagy.