PMC:4996362 / 11871-13817 JSONTXT

{"project":"2_test","denotations":[{"id":"27600341-17308349-69477249","span":{"begin":613,"end":615},"obj":"17308349"}],"text":"3.2. Immunocharacterization of AF22 iPS-Derived Cells by CMA Technology versus Cover Slip\nAF22 iPS-derived cell line was induced to differentiate into neuronal lineage for 21 days in three different methodological conditions (coverslip, trypsinization and mechanical scraping as described in the material and method section). Agarose cell pellets were embedded in paraffin and CMA construction was assessed using the Galileo CK4500 platform. IF analysis was performed to investigate the expression and distribution of the stem cell markers Nestin, Sox 2, SEL1L and β3‑tubulin, as previously described (Figure 3) [17]. The mechanical harvesting (3D) of the cells facilitates the immunofluorescence analysis when compared to the enzymatic treatments (3C). Detachment of the cells using a rubber policeman gathers the majority of the cells from the flask maintaining the number of cells and preserving the elongated neuronal morphology. Moreover, this approach allows better marker localization (more similar to coverslip grown cells) if compared to trypsinized cells. These characteristics are very suitable for CMA applications.\nFigure 3 Immunocharacterization of the AF22 iPS-derived cell line by CMA technology versus coverslip. Undifferentiated (A) and differentiated AF22 cells (B) grown on coverslip were analyzed for Nestin, Sox2 and β3-tubulin expression by immunofluorescence. Undifferentiated cells were uniformly immunopositive for the neural precursor cell markers Nestin, Sox2 and SEL1L, a protein involved in neural lineage commitment but negative for the neuronal differentiation markers β3-tubulin. A clear decrease of Nestin, Sox2 and SEL1L is shown in the differentiated (B) cells with the concomitant increase of β3-tubulin expression. AF22 cells grown in stemness and differentiated conditions were simultaneously grown in flasks and collected either by trypsinization (C) or with a rubber policemen (D). Scale bars: 100 µm.\n\n4"}