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AN OPTIMIZED METHOD TO DIFFERENTIATE HL60 CELLS INTO NEUTROPHIL-LIKE CELLS
Samuel P Collie1, Grant Yu2, Suraj S Rawat3
1Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, MI.
Biorxiv : the Preprint Server for Biology
|January 16, 2026
Summary
Researchers developed a new method to create neutrophil-like cells from HL60 leukemia cells. This improved protocol yields cells that better mimic mature human neutrophils in form and function.
Area of Science:
- Hematology
- Cell Biology
- Immunology
Background:
- The HL60 promyelocytic leukemia cell line is a valuable tool for studying neutrophil biology.
- Current differentiation methods using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO) yield neutrophil-like cells lacking key mature granulocyte features.
- ATRA treatment results in poor chemotaxis, while DMSO treatment leads to abnormal nuclear morphology.
Purpose of the Study:
- To develop an improved protocol for differentiating HL60 cells into neutrophil-like cells.
- To generate HL60-derived neutrophils that more accurately replicate the morphology and functionality of primary human neutrophils.
Main Methods:
- A sequential differentiation protocol was established: one day of ATRA treatment followed by four days of DMSO treatment (A1D4).
- Morphological analysis focused on nuclear segmentation and lamin A/C expression.
- Functional assays included chemotaxis, oxidative burst, and phagocytosis.
Main Results:
- The A1D4 protocol produced HL60-derived neutrophils with segmented nuclei and reduced lamin A/C expression, characteristic of mature neutrophils.
- These cells exhibited robust chemotactic, oxidative burst, and phagocytic activities.
- The A1D4 method successfully combined the benefits of ATRA and DMSO treatments.
Conclusions:
- The sequential A1D4 differentiation protocol significantly enhances the fidelity of HL60 cells as neutrophil-like models.
- This method overcomes the limitations of single-agent treatments, providing a more accurate representation of primary neutrophil phenotype and function.
- A1D4-differentiated HL60 cells offer a superior tool for research in neutrophil biology and related fields.
