PMC:4996362 / 4607-7329
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996362","sourcedb":"PMC","sourceid":"4996362","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996362","text":"2.1. Cell Culture for CMA Preparation\nThe CMA construction requires the initial preparation of cell paraffin blocks [12]. Human AF22 induced pluripotent stem (iPS) cell-derived long term neural stem (lt-NES) cells [13] were cultured according to previously described method [14]. Briefly, cells were maintained in 0.01% Poly-L-ornithine (Sigma, Milan, Italy) and 10 μg/mL laminin (Sigma) coated flask, using DMEM-F/12 medium (Euroclone, Milan, Italy) supplemented with 1% N2 and 0.1% B27 (Life Technologies, Monza, Italy), 10 ng/mL EGF and 10 ng/mL FGF2 (Peprotech EC, London, UK). To induce neuronal differentiation, cells were plated in Neurobasal (Life Technologies) and DMEM-F/12 (Euroclone) 1:1 with 0.5% N2, 1% B27, without EGF and bFGF and cultured for 3 weeks. Cells were passaged every 2 to 3 days using 0.05% trypsin-EDTA solution (Sigma). \nThree testing conditions were used for array construction: (i) cell trypsinization, (ii) mechanical by scraping with a rubber policeman and (iii) growth on coverslips.\n(i) Cell trypsinization: The cells, 5 × 106, were detached from the plate using trypsin-EDTA (Sigma), washed several times with PBS (Euroclone), and the final cell pellet was fixed in 4% PFA (Electron Microscopy Sciences) at 4 °C for 20 min. The pellet thus formed was resuspended in 300 μL of low melting agarose 3% (Sigma), in 1× PBS at 55 °C and incubated for 30 min on ice. After 24 h incubation in 1% PFA, prior to the paraffin embedding procedure, the agarose pellet was processed in an automatic tissue processor (Fully Enclosed tissue Processor Leica ASP300S, Leica Microsystems, Milan, Italy) for dehydration by incubating the pellet in solutions with increasing alcohol content under vacuum pressure. \n(ii) Mechanical Scraping: About 5 × 106 were immediately fixed in 4% paraformaldehyde (PFA) for 20 min, scraped with a rubber policeman and centrifuged 3 min at 300 × g at 4 °C. The fixed cells were gently re-suspended in 300 μL of low melting agarose (3%) in 1× PBS at 55 °C, transferred into a 0.5 mL conical tube and incubated in ice for 30 min. The cell-agarose-pellet was fixed in 1% PFA for 24 h and then embedded in paraffin with an automatic tissue processor. To evaluate an optimal cell concentration and density, 5 μm sections were hematoxylin-eosin stained and were used to select appropriate areas for CMA construction. \n(iii) Cells grown on coverslip: AF22-iPS derived cells were grown on small, circular coverslips and placed in wells of a tissue culture plate. After fixation, the cells on coverslips were removed from the wells of the plate for antibody staining [15]. Grown cells were fixed in cold 4% paraformaldehyde for 15 min at room temperature (RT) and washed in PBS.\n","divisions":[{"label":"Title","span":{"begin":0,"end":37}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600341-16957166-69477243","span":{"begin":117,"end":119},"obj":"16957166"},{"id":"27600341-22272239-69477244","span":{"begin":215,"end":217},"obj":"22272239"},{"id":"27600341-19218428-69477245","span":{"begin":275,"end":277},"obj":"19218428"},{"id":"27600341-18989438-69477246","span":{"begin":2611,"end":2613},"obj":"18989438"}],"attributes":[{"subj":"27600341-16957166-69477243","pred":"source","obj":"2_test"},{"subj":"27600341-22272239-69477244","pred":"source","obj":"2_test"},{"subj":"27600341-19218428-69477245","pred":"source","obj":"2_test"},{"subj":"27600341-18989438-69477246","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93a8ec","default":true}]}]}}