HaCaT cells were purchased (HDFa lot #1780051, Gibco, United States) and expanded in Dulbecco’s modified eagle medium (DMEM; Gibco) containing 8% fetal bovine serum. Skin mast cells (Kim et al., 2018 (link)) and dermal fibroblast cells (Seluanov et al., 2010 (link)) were isolated according to the standard procedures. For isolation of mouse skin dermal fibroblast cells, the underarm area of Nc/Nga mouse was shaved and cut off and the tissue fragments were transferred to culture dish. Tissue fragments were cut into 1 mm pieces and incubated for 1 h at 37°C with liberase TM solution (Sigma). After digestion, DMEM/F12 medium was added to stop liberase digestion. Centrifugation to remove liberase was followed and the pellet was resuspended in DMEM/F12 media. After washing, the final pellet was resuspended in media and incubation was continued on cell plates for 7 days. Fibroblasts start to exit from tissue fragment within 5 days. Dermal fibroblasts at passage four or five were used in this study.
Liberase solution
Manufactured by Merck Group
Sourced in United States
Liberase TM solution is a laboratory-grade enzyme blend designed for the dissociation and isolation of cells from various tissue types. The solution contains a proprietary mixture of proteolytic enzymes that effectively break down the extracellular matrix, facilitating the release of viable cells. The core function of Liberase TM solution is to enable the efficient extraction and preparation of cells from complex tissue samples for further analysis or experimentation.
Automatically generated - may contain errors
2 protocols using liberase solution
1
Isolation and Culture of Skin Cell Types
2
Isolation of Primary Mouse Hepatocytes and Liver Mononuclear Cells
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Primary mouse HCs and liver mononuclear cells (MNCs) were isolated as previously described 11 (link) . Brie y, mouse livers were perfused with liberase TM solution (Sigma-Aldrich, St. Louis, MO, USA) after euthanasia, ltered through a 70 μm nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA), and centrifuged at 20× g for 5 min. The pellets were suspended and placed on the surface of 30% Percoll solution, centrifuged at 1000 × g for 10 min at 4 °C and washed once with phosphate-buffered saline (PBS). HCs were used for mRNA/miRNA isolation or protein extraction. Supernatants containing MNCs were collected, resuspended in 30% Percoll, and gently overlaid onto 70% Percoll. After centrifugation at 1000 × g for 30 min, liver MNCs were harvested from the interphase, washed twice with PBS, and then resuspended for further uorescence-activated cell sorting (FACS) analysis.
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