The specimens without adipose layers that were cut into pieces by scissors, the epidermis and dermis were separated by treatment with 0.25% dispase enzyme (Sigma Aldrich, St. Louis, MO, USA). The dermis was minced finely and turned into a single cell suspension. Cells were then cultured in DMEM/F12 (HyClone, USA)containing 10% FBS (HyClone, USA), 10 μl/ml B27 (Invitrogen, US), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37℃ with 5% CO2. The non-adherent cells were removed after 48 h, and the remaining cells were cultured with passaging. DMSCs were identified using flow cytometry to analyse CD29 (Backman, USA), CD44 (Backman, USA), CD105 (Backman, USA), CD34 (Backman, USA), CD45 (Backman, USA), HLA-DR (Backman, USA), Dil-LDL (Sigma-Aldrich, St. Louis, MO, USA), and UEA-1-FITC (Sigma-Aldrich, St. Louis, MO, USA). Iden-tification was also performed on differentiated adipocytes, osteoclasts and VECs. The specific methods followed published protocols (13 (link), 14 (link)).
Dispase enzyme
Manufactured by Merck Group
Sourced in United States
Dispase enzyme is a neutral protease derived from Bacillus polymyxa. It is a broad-spectrum enzyme capable of hydrolyzing various components of the extracellular matrix, including collagen, fibronectin, and laminin. The enzyme is commonly used in cell and tissue dissociation applications, such as the isolation of primary cells from tissues.
Automatically generated - may contain errors
2 protocols using dispase enzyme
1
Isolation and Characterization of Dermal Mesenchymal Stem Cells
2
Long-term Organoid Self-Renewal Assay
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To study long-term self-renewing potential, organoids were dissociated and re-plated for the next passage. Prior to passaging, Matrigel was dissolved by incubation with dispase enzyme (1 mg/mL for 30 min at 37°C, Sigma), and organoids with a size greater than 50 μm were counted using an ocular micrometer with a 10× objective. Initial parathyrosphere cultures 7 days of age were dissociated to single cells using 0.05% trypsin-EDTA (Invitrogen) and counted using trypan blue, and cell concentration was adjusted to 2 × 106 cells/mL. In total, 20 μL of this cell solution was combined on ice with 40 μL of Basement Membrane Matrigel (BD Biosciences) and plated in the center of a 24-well tissue culture plate. After solidifying the Matrigel for 30 min at 37°C, gels were covered in 500 μL of parathyroid medium as defined above, and parathyroid medium was renewed weekly. This self-renewal procedure was repeated every 3–4 weeks and up to 5 times (4 passages).
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