Induced mesenchymal progenitor cells (iMPCs) were generated from iPSCs using a spontaneous differentiation protocol as described before [26 (link),32 (link)]. When iPSCs reached 70~80% confluency, the iPSCs’ maintenance medium was switched to STEMdiffTM-ACF Mesenchymal Induction Medium (Stemcell Technologies, Vancouver, BC, Canada) for three days, and the medium was changed every day afterwards. The medium was subsequently replaced by MesenCultTM-ACF Plus Medium (Stemcell Technologies) for four days of differentiation. The cells were detached and plated onto pre-coated plates within the MesenCultTM-ACF Plus Medium. When the cultures reached 70~80% confluency, ACF Enzymatic Dissociation Solution and ACF Enzyme Inhibition Solution (Stemcell Technologies) were used for detachment; the differentiated cells were then grown in tissue culture flasks (Corning, NY, USA) for future experiments. The iMPCs were cultured in the fibroblast differentiation medium for four weeks as described above for MSCs before use.
Acf enzymatic dissociation solution
Manufactured by STEMCELL
Sourced in United States, Canada
ACF Enzymatic Dissociation Solution is a cell dissociation reagent designed for the gentle and effective dissociation of cells from various tissue types. It contains a proprietary blend of enzymes that facilitate the breakdown of the extracellular matrix, allowing for the release of individual cells while maintaining high cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Acf enzyme inhibition solution, by STEMCELL (2 mentions)
Stemdiff acf mesenchymal induction medium, by STEMCELL (2 mentions)
Mesencult acf plus medium, by STEMCELL (2 mentions)
Tissue culture flask, by Corning (1 mentions)
Pneumacult ex plus media, by STEMCELL (1 mentions)
Bepicm, by ScienCell (1 mentions)
Poly l lysine (pll), by ScienCell (1 mentions)
Antibiotics antimycotic, by Thermo Fisher Scientific (1 mentions)
Animal component free cell attachment substrate, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
4 protocols using acf enzymatic dissociation solution
1
Generation of iMPCs from iPSCs
2
Isolation and Differentiation of Human Nasal Epithelial Cells
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Detailed protocols for the isolation, growth and differentiation as well as immunohistochemistry of human nasal epithelial cells can be found in Manna and Caradonna [20] . Human nasal epithelial cells (HNECs) were isolated through brushing of inferior and middle nasal turbinates with interdental brushes (GUM). Brushes were removed from the handle and placed into an enzymatic digestion buffer (ACF Enzymatic Dissociation Solution, Stem Cell Technologies), briefly vortexed followed by incubation at 37 °C for 15 min. After digestion cells were pelleted through centrifugation at 200 x g for 5 min, pellets were suspended in basal cell expansion media (PneumaCult Ex Plus Media, Stem Cell Technologies) and incubated at 37 °C, 5%CO2. Culture media was refreshed every two days, HNECs were collected and counted between 8 and 10 days of culture. HNECs were then either seeded directly onto transwell inserts for ALI differentiation or banked in liquid nitrogen for future experiments. Protocols for isolation of HNEC specimens from human subjects is approved by our Institutional Review Board and all donors provided informed consent.
3
Cigarette Smoke Exposure in Human Bronchial Epithelial Cells
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Human bronchial epithelial cells (HBEpiCs/HBECs, ScienCell and Lonza/Fischer Scientific) were cultured in BEpiCM (ScienCell) at 37 °C in an atmosphere containing 5% CO2 until 80–90% confluence. Next, cells were washed once with PBS, and treated with trypsin (ACF Enzymatic Dissociation Solution, Stemcell Technologies, Vancouver, Canada) followed by incubation at 37 °C in 5% CO2 for 5 min. Once the cells were dissociated, an equal volume of trypsin solution was added, followed by centrifugation at 230 × g for 5 min before aspirating the medium and re-suspending the cell pellet in 1 mL fresh medium. For experiments, 24-well plates were coated with poly-L-Lysine (ScienCell) for at least 1 h at 37 °C. Wells were washed once with Dulbecco’s PBS before seeding the cells (30 000 cells/well). Upon reaching 80% confluence, cells were stimulated with 5% (v/v) CSE for 24 h at 37 °C. At the end of the incubation period, plates were centrifuged at 230 × g for 5 min before collecting cells and supernatants for further experiments.
4
Efficient iMPC Generation from iPSCs
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iMPCs were generated from iPSCs using a protocol established in our lab (Diederichs and Tuan, 2014 (link)). When iPSCs reached 80% confluency, expansion medium was replaced with STEMdiff™-ACF Mesenchymal Induction Medium (Stemcell Technologies) for 3 days. Afterwards, MesenCult™-ACF Plus Medium (Stemcell Technologies) was used. On day 6, differentiated cells were detached and re-plated onto flasks that were pre-coated with Animal Component-Free Cell Attachment Substrate (Stemcell Technologies). The MesenCult™-ACF Plus Medium was used to expand these cells to 80% confluency. After being dissociated with ACF Enzymatic Dissociation Solution and ACF Enzyme Inhibition Solution (Stemcell Technologies), the iMPCs were grown on regular tissue culture flasks in expansion medium [DMEM/F-12 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), and 1% antibiotics-antimycotics (Gibco)]. iMPCs at passage 4 were used for all experiments.
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