Journal: Cells

Article Title: Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells

doi: 10.3390/cells11091379

Figure Lengend Snippet: ( A ) After Nycodenz separation, high-lipid-content cells float (white ring) between the Nycodenz solution and the GBSS + NaCl solution (yellow arrow). ( B ) Cell viability assessed with fixable EF 780 viability dye back gated in side scatter (SSC) and forward scatter (FSC)-contour plot with outliers as dots. Green: viable cells; red: dead cells. Proportion expressed as a mean ± SD, n = 3. ( C , D ) Quantitative polymerase chain reaction assessing DCN (coding for Decorin) ( C ) and CLEC4F (coding for Clefc4f) ( D ) gene expression in cell populations after the two major steps of HSC isolation (Nycodenz and MACS sorting). Values obtained were expressed as fold-increase (FI) where the value obtained by non-parenchymal cell (NPC) population was set as 1. Decorin has been described to be specific to HSCs whereas CLEC4F is expressed by Kupffer cells. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.00, if not specified, non-significant. n = 2.

Article Snippet: Nycodenz density gradient solution was from Axon lab AG (Le Mont-sur-Lausanne, Switzerland).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Isolation

Journal: Cells

Article Title: Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells

doi: 10.3390/cells11091379

Figure Lengend Snippet: Cell yield per mouse after HSC Nycodenz separation alone or after additional negative CD11b selection. Nycodenz: yield without CD11b negative selection. CD11b−: HSC enriched fraction (flow through). CD11b+: macrophages (selected cells). SEM: standard error of the mean. Yield variations reflected fluctuations in liver digestion and learning curve of the isolation procedure. n = 3 to 7.

Article Snippet: Nycodenz density gradient solution was from Axon lab AG (Le Mont-sur-Lausanne, Switzerland).

Techniques: Selection, Isolation

Journal: Scientific Reports

Article Title: A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells

doi: 10.1038/s41598-019-49287-7

Figure Lengend Snippet: Schematic overview of the protocol for isolation of human HSC. ( a ) Surgically removed fresh normal liver tissues were processed and collected in an ice box at 4 °C under aseptic conditions. ( b ) The connective tissues, bile ducts, and blood vessels were trimmed away as much as possible using ophthalmic scissors, and the tissue were cut into small approximately 1-mm pieces. ( c ) Gentle MACSTM tissue processor disrupt the tissue in a gentle and stable setting. ( d ) Stop digestion and filtered. ( e ) Isolation human hepatic cells and human hepatic stellate cells. ( f ) Nycodenz density gradient centrifugation.

Article Snippet: The aliquots were cryopreserved at −20 °C. (Miltenyi Biotec, 130-093-237, Germany) DNase I (Roche, Switzerland) Nycodenz Separation Solution (density 1.131 g/ml, Beijing Solarbio Science & Technology Co., Ltd.) RPMI 1640 medium (Gibco, USA) DMEM/F12 medium (Gibco, USA) Gey’s balanced salt solution (GBSS, Sigma, USA) Phosphate-buffered saline (PBS, Gibco, USA) Penicillin-streptomycin (Gibco, USA) Fetal bovine serum (FBS, Gibco, USA) Oil red O (Sigma, USA) Trypan blue (Beyotime Biotechnology, China) Primary antibodies: Rabbit anti-α-smooth muscle actin (SMA) monoclonal antibody (Abcam, USA) Secondary antibody: goat anti-rabbit IgG (Abcam, UK).

Techniques: Isolation, Gentle, Gradient Centrifugation

Journal: Scientific Reports

Article Title: A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells

doi: 10.1038/s41598-019-49287-7

Figure Lengend Snippet: ( a ) The solution was filtered through a SmartStrainer filter. ( b ) Isolate the hepatocytes. ( c ) The pellet containing the HSCs. ( d ) DMEM washing pellet. ( e ) Preparation of 15 ml tubes for Nycodenz density centrifugation. ( f ) After density centrifugation, HSCs can be identified as a white cell layer that is floating at the surface of the gradient. ( g ) The HSC-containing layers are collected and combined, filled with GASS, and centrifuged again. After centrifugation, a white pellet is visible.

Article Snippet: The aliquots were cryopreserved at −20 °C. (Miltenyi Biotec, 130-093-237, Germany) DNase I (Roche, Switzerland) Nycodenz Separation Solution (density 1.131 g/ml, Beijing Solarbio Science & Technology Co., Ltd.) RPMI 1640 medium (Gibco, USA) DMEM/F12 medium (Gibco, USA) Gey’s balanced salt solution (GBSS, Sigma, USA) Phosphate-buffered saline (PBS, Gibco, USA) Penicillin-streptomycin (Gibco, USA) Fetal bovine serum (FBS, Gibco, USA) Oil red O (Sigma, USA) Trypan blue (Beyotime Biotechnology, China) Primary antibodies: Rabbit anti-α-smooth muscle actin (SMA) monoclonal antibody (Abcam, USA) Secondary antibody: goat anti-rabbit IgG (Abcam, UK).

Techniques: Centrifugation