Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

Figure 2

Gating strategy for the purification of hepatic stellate cells using fluorescence-activated cell sorting. Cells are first gated based on their forward and sideward scattering (a), doublets are excluded from sideward (b) and forward scattering (c), and hepatic stellate cells (HSC) are selected based on the UV light excitation of retinol (vitamin A) (d). A detailed cell-type specific staining of the Kupffer cell marker F4/80 demonstrated that the large (here: FSC-A > 100 in the plot, dotted gate) retinol⁺ cells are Kupffer cell- (KC-) HSC doublets that stain positive for F4/80 (right hand side), whereas the smaller retinol⁺ cells are F4/80⁻ HSC (left hand side). By placing a sorting gate as depicted in (d) (middle plot, black line), selected HSC (left plot) are pure and do not contain contaminating KC.