Journal: Analytical cellular pathology (Amsterdam)
Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells
Figure Lengend Snippet: Hepatic stellate cell functionality in vitro . Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol and fluorescence-activated cell sorting (FACS). The HSC were cultured in DMEM with 10% fetal calf serum, and some plates were stimulated with lipopolysaccharides (LPS) at 100 ng/mL (after five days of culture) for another 48 hours. Changes in the cell number during culture were determined from time lapse microscopy (a) and statistical summary (b). HSC were cultured for five days on tissue culture-treated polystyrene in DMEM with 10% fetal calf serum including culture inserts for self-insertion (“scratch assay”). To start horizontal migration, the plastic inserts were removed and HSC migrated (c) and were quantified using software (d). HSC were cultured for designated periods and quantitative real-time PCR was performed to study the expression of α smooth muscle actin ( α SMA), collagen 1 (Col1A1), or the transforming growth factor β (TGF β ) as markers of HSC activation. Gene expression was normalized to β -actin expression of cells that were lysed directly after isolation at day zero (e). Mean ± SD of three independent experiments.
Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).
Techniques: In Vitro, Isolation, Mouse Assay, Gradient Centrifugation, Fluorescence, FACS, Cell Culture, Time-lapse Microscopy, Migration, Software, Real-time Polymerase Chain Reaction, Expressing, Activation Assay