0.02 mg/ml) for 15 min at room temperature. Specimens were analyzed using a LEICA DMR fluorescence microscope. Images were analysed using Image J software. For the analysis lipid loss from LDs during the activation process, freshly isolated HSCs were incubated with 25 mM Bodipy 558/568-C12 for 5 h at 37uC. After washing to remove the excess of fluorescent lipid, cell culture was continued, and cells were fixed with 4% paraformaldehyde at several time points. To study the dynamics of lipid incorporation into LDs during the HSC activation process, cells at day 0, 4 and 7 were incubated with 25 mM Bodipy 558/568-C12 for 5 h at 37uC and fixed with 4% paraformaldehyde. Specimens were analyzed using a LEICA DMR fluorescence microscope. For live cell imaging, cells were cultured on Fluorodisk wells and placed in a Solent scientific cell incubator with temperature and CO2 control. Life cell analysis was performed for 12 hours from 72 h to 84 h after plating Time lapse series were obtained using a BioRad Radiance 2100 MP confocal system (Zeiss/BioRad, Hertfordshire, UK). Confocal Raman microspectroscopy and imaging Freshly isolated HSCs were grown on calcium fluoride cover slips and fixed at day 0, 4 and 7 with 2% paraformaldehyde in PBS for 15 minutes. In some experiments cells at day 0, 3 and 6 were incubated with 25 mM deuturated arachidonic acid (20:4-d8) for 24 h, whereafter they were fixed. For calibration of the RE and TAG detection pure lipid standards (retinyl palmitate and trioleylglycerol) and mixtures of several ratios hereof were used. Non-resonant Raman spectroscopy and imaging experiments were performed on a laser-scanning confocal Raman microspectrometer [13]. Imaging experiments were performed by raster-scanning the laser beam over a LD or an intracellular region of interest and accumulating a full Raman spectrum at each pixel (1 s/pixel at 23 mW 647.1-nm excitation power). Noise in the resulting 3D (spatial6spatial6spectral dimension) data matrix was reduced by singular value decomposition [13,16]. Raman images were constructed by plotting the integrated intensity of the vibrational band of interest as a function of position. Hierarchical cluster analysis (HCA) was performed on Raman imaging data matrices to visualize regions in cells with high Raman spectral similarities. In the cluster analysis routine, principal component analysis scores were taken as input variables, squared Euclidean distances were used as distance measure, and Ward’s algorithm was used to partition Raman spectra into clusters. All data manipulations were performed in routines written in Matlab 6.5 (MathWorks, Natick, MA). High-performance liquid chromatography and mass spectrometry Samples were kept under red light and all procedures were carried out in brown vials to prevent retinoid isomerization and oxidation. Lipids were extracted from equal protein amounts of total cell homogenate of HSC at day 0, 4, 7 and 1 month after isolation by the method of Bligh and Dyer [39]. Extracted lipids were separated in a neutral and phospholipid fraction by fractionation on a freshly prepared silica-G column (approximately 10 mg of 0,063–0,200 mm silica) [40]. Lipid extracts were dissolved in methanol/chloroform (1/9, v/v) and loaded on top of the silica column. Neutral lipids were eluted with two volumes acetone, dried under nitrogen gas and stored at 220uC. Just before HPLC-MS analysis, the neutral lipid fraction was reconstituted in methanol/chloroform (1/1, v/v) and separated on a Lichrospher RP18-e column (5 mm, 25064.6 mm; Merck, Darmstadt, Germany). A gradient was generated from acetonitrile to acetone/chloroform 85/15, v/v, at a constant flow rate of 1 ml/min. Mass spectrometry of lipids was performed using Lipidome of Activated Hepatic Stellate Cells Atmospheric Pressure Chemical Ionization (APCI) on a Biosys- tems API-4000 Q-trap (MDS Sciex, Concord, Canada). The system was controlled by Analyst version 1.4.2 software (MDS Sciex, Concord, ON, Canada) and operated in positive ion mode using the following settings: source temperature 420uC, nebulizer gas (GS1) 5, nebulizer current 3 mA, curtain gas 10, collision gas High and declustering potential 100 V. The optimal collision energy was dependent on the type of experiment and was set to +10 V (full scan mode) or +30 V (product ion mode). In all full scan runs spectra were obtained from m/z 250–1100. To identify the exact composition of high m/z value TAG species, an enhanced product ion spectrum (optimal CE +35 V) was made of all ions with an m/z value over 850. The solvents and gradient used were similar as described above. The phospholipid fraction was dissolved in methanol/acetoni- trile/chloroform/water (46:20:17:17) and directly injected into the mass spectrometer. Mass spectrometry of phospholipids was performed using Electrospray Ionization (ESI) on a Biosystems API-4000 Q-trap. The system was controlled by Analyst version 1.4.2 software and operated in positive mode (phosphatidylcholine (PC), precursor scan of 184 m/z and phosphatidylethanolamine (PE), neutral loss of 141 amu) and negative mode (phosphatidy- linositol (PI), precursor scan of 241 m/z and phosphatidylserine (PS), neutral loss of 87 amu), using the following settings: source temperature 450uC, nebulizer gas (GS1) 45, curtain gas 10 and collision gas High. Declustering potential and collision energy were depending on the type of experiment and were respectively for PC 120 V and 47 V, for PE 110 V and 30 V, for PI 2110 V and 260 V and for PS 2100 V and 236 V. Data analysis was performed using Analyst 1.4.2 software (MDS Sciex, Concord, ON, Canada) and calibration curves of all lipid classes were established under similar conditions as the samples. Supporting Information Figure S1 Migration of perinuclear LDs towards the growing cell extensions in activated HSCs. To investigate LD redistribution during HSC activation, freshly isolated HSCs were incubated at day 0 with 25 mM Bodipy C-12 for 5 h. After washing the excessive Bodipy C-12, cells were fixed (day 0) or cultured for 4 days in the absence of dye before fixation (day 4). Cells were analyzed by fluorescence microscopy. (TIF) Figure S2 Changes in protein levels of various enzymes implicated in LD metabolism during HSC activation. Western blots of equal amounts of total protein from isolated rat HSC one day after plating (dy1) and 7 days after plating (dy7). Blots were probed with antibodies against CTP:phosphocholine cytidylyltransferase alpha (CCTa), diacylglycerol acyltransferase 2 (DGAT2), lecithin:retinol acyltransferase (LRAT), and b-actin (ACT; loading control). (TIF) Figure S3 Incorporation of PUFAs in phosphatidylcho- line species is not increased during HSC activation. Phospholipid extracts of HSCs harvested at day 0, 4 and 7 were analyzed by HPLC-MS as described. The results represent the means 6 SEM of three experiments. (TIF) Movie S1 LD dynamics in activated HSC. Time lapse life cell analysis was performed for 12 h on a typical HSC from 72 h to 84 h after plating as described in method section. Frames were taken every 30 minutes. (AVI) PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34945