Ck 19

A liver cell suspension was prepared using a 2-step collagenase perfusion technique with modifications as described previously.³³ (link)^,34 In brief, hepatocytes were removed by multiple cycles of low-speed centrifugation, which resulted in a supernatant that contained the nonparenchymal cells. phHSCs were subsequently purified from the nonparenchymal cell fraction by discontinuous density centrifugation using Percoll.³³ (link) Quality of isolation was checked using immunocytomicrocopy against α-SMA (Sigma-Aldrich), Desmin (Sigma-Aldrich), GFAP (Sigma-Aldrich), and CK-19 (Merck Millipore) (Figure 14).Figure 14

Characterization of isolated phHSCs. Representative immunocytomicroscopy figures showing that more than 95% of isolated cells exhibit typical markers of HSCs, including α-SMA, GFAP, and Desmin.

Cells were stimulated for 24 hours with 1000 nM CEP-1347 before undergoing incubation with Hoechst 33342 (Fluka, Buchs, Switzerland). For immunocytomicroscopy following antibodies were used: α-SMA (Sigma-Aldrich), p70S6K (Cell Signaling Technology), phospho-p70S6K (Cell Signaling Technology), CK-19 (Merck Millipore), Desmin (Sigma-Aldrich), and GFAP (Sigma-Aldrich). As secondary antibody, an Alexa-Fluor 488 goat anti-mouse IgG or goat anti-rabbit IgG was used. Hoechst or phalloidin (Invitrogen) were used for counterstaining. Microscopy was performed using a Zeiss Axiovert TV 135 (Zeiss, Oberkochen, Germany).