PMC:4996362 / 4582-8942 JSONTXT

{"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. Experimental Section\n\n2.1. 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\n2.2. CMA Construction\nGalileo TMA CK4500 platform (Isenet, Milan, Italy) was used for CMA construction. A hollow needle of 1mm diameter was used to pick cell cores from the donor paraffin block, which were then assembled in the recipient block. Up to 80 consecutive sections of 5 µm thickness can be cut from the CMA block and mounted on microscope slides. The slides were now ready to be assayed with different stemness and differentiation markers.\n\n2.3. Immunofluorescence (IF) Analysis\nCell array slices were deparaffinized and rehydrated by incubating in solutions with decreasing alcohol content. Antigen retrieval was conducted by boiling the sample slides in citrate buffer 0.01 M pH 6. Both coverslip and CMA slices were incubated in 0.1 M glycine for 10 min at RT and then in blocking solution composed by 5% donkey serum, 0.6% Triton in PBS for 30 min at RT. Samples were immunostained at 4 °C overnight in blocking solution with primary antibodies anti-Nestin (1:50; Millipore, Milan, Italy), anti-sox2 (1:300; Millipore), anti-β3-tubulin (1:100; Sigma) and anti-sel1L (5 μg/mL) [16]. For the immunofluorescence characterization, samples were incubated with appropriate secondary antibodies (Rhodamine-Red antimouse IgM and anti-rabbit IgG, Alexa Fluor 488 anti-mouse IgG, (Jackson ImmunoResearch, distributed by Li StarFish, Milan, Italy) and the nuclei were counterstained with Hoechst 33258. The samples were mounted with GelMount aqueous mounting medium (Sigma). The images were acquired using a Leica DMI4000B inverted microscope linked to a DFC360FX or to a DFC280 cameras (Leica Microsystems).\n","divisions":[{"label":"Title","span":{"begin":0,"end":23}},{"label":"Section","span":{"begin":25,"end":2747}},{"label":"Title","span":{"begin":25,"end":62}},{"label":"Section","span":{"begin":2748,"end":3198}},{"label":"Title","span":{"begin":2748,"end":2769}},{"label":"Section","span":{"begin":3199,"end":4359}},{"label":"Title","span":{"begin":3199,"end":3236}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600341-16957166-69477243","span":{"begin":142,"end":144},"obj":"16957166"},{"id":"27600341-22272239-69477244","span":{"begin":240,"end":242},"obj":"22272239"},{"id":"27600341-19218428-69477245","span":{"begin":300,"end":302},"obj":"19218428"},{"id":"27600341-18989438-69477246","span":{"begin":2636,"end":2638},"obj":"18989438"},{"id":"27600341-12113576-69477247","span":{"begin":3839,"end":3841},"obj":"12113576"}],"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"},{"subj":"27600341-12113576-69477247","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93c5ec","default":true}]}]}}