Protocol to isolate quiescent hepatic stellate cells in sufficient yield while maintaining cytokine responsiveness from aged female mice

Isolation of primary hepatic stellate cells (HSCs) in sufficient quantities is technically challenging. We present a protocol for isolating quiescent HSCs from mice using Nycodenz density-gradient centrifugation. The procedure includes cervical dislocation, vena cava cannulation, liver perfusion, enzymatic digestion, and preparation for density separation. We then describe centrifugation, enrichment, washing, counting, and culture preparation. This protocol provides fresh HSCs suitable for diverse experimental applications. For complete details on the use and execution of this protocol, please refer to Seki et al.¹.