Under what circumstances is cell sorting required, and how should the appropriate method be selected?

Answer:
Cell sorting is recommended under the following conditions:

• The initial proportion of target cells is low (typically less than 8%), or the absolute number of target cells is insufficient for downstream analysis. 
• The study focuses exclusively on a specific cell population. 
• Certain cell types need to be removed (e.g., neutrophils, dead cells, or red blood cells). 

Before proceeding with large-scale experiments, it is strongly recommended to perform a pilot experiment to confirm that the sorted cell suspension meets the required quality criteria, including cell viability (>90%), aggregation rate (<10%), nucleated cell proportion (>70%), and adequate cell concentration.

Two primary sorting strategies are commonly used:

• Fluorescence-activated cell sorting (FACS): allows simultaneous selection using multiple markers and typically provides higher specificity and purity. 
• Magnetic bead-based sorting: is easier to operate and offers higher throughput, but may have lower specificity compared to FACS. 

Several technical considerations should be noted:

• Technical bias: Sorting is based on protein-level marker expression, whereas single-cell sequencing measures transcript-level expression. Discrepancies between these two levels may result in differences between expected and observed cell populations. 
• Resuspension buffer: Cells should be resuspended in an appropriate buffer compatible with downstream applications. Buffers containing high protein content or incompatible additives may affect cell viability or interfere with downstream reactions.
• Cell quality control: Post-sorting quality assessment is critical. In addition to viability and aggregation, attention should be paid to debris levels and potential enrichment of damaged cells. Poor-quality sorted samples may lead to increased background and reduced data quality.
• Fluorescence compatibility: Fluorophores used during sorting should not overlap with dyes used for cell counting (e.g., AOPI), as this may affect counting accuracy.
• Sorting-induced stress: Prolonged sorting procedures, especially FACS, may reduce cell viability and alter gene expression profiles.

Magnetic bead-based sorting is widely used due to its simplicity and scalability; however, several factors may affect sorting efficiency and purity.

• When the proportion of target cells is lower than approximately 8%, or when the total number of target cells (total cell number × target cell proportion) is less than 100,000, sorting efficiency may be reduced. In such cases, performing two rounds of magnetic bead sorting is recommended to improve enrichment efficiency.  
• If the sample exhibits a high aggregation rate (e.g., greater than 20%), cell clusters may become trapped within the sorting column. This can lead to blockage and may result in contamination of the positively selected fraction with non-target cells.  
• Proper sample dissociation and filtration prior to sorting are therefore critical to ensure optimal performance.