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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Nat Protoc. 2015 Jun 11;10(7):985–1006. doi: 10.1038/nprot.2015.065

Figure 5. Analysis of soluble Aβ levels in the enriched FAD ReN cells by Western Blot and Aβ ELISA.

Figure 5

a. Western Blot of Aβ levels in the conditioned medium collected from undifferentiated enriched ReN-G and -GA cells. The conditioned medium was collected from T25 flask after culturing the cell for 2 days. 6E10 anti-Aβ detected ~4 kDa Aβ monomer bands in the media collected from the enriched FAD ReN cells (ReN-GA10, ReN-GA2). The 6E10 antibody also detected sAPPα (~100 kDa), which would represent the amount of full-length APPs in these cell lines. b. Increased Aβ40 and 42 levels in undifferentiated FAD ReN-GA2 cells, detected through Aβ ELISA (***, p<0.001; Student t-test; n=3 per each sample). c. Increased Aβ40 and 42 levels were also detected in a conditioned media collected from 1-week old thick-layer 3D cultures of ReN-GA2 cells (***, p<0.001; Student t-test; n=3 per each sample). The soluble Aβ levels cannot be directly compared to the levels shown in (b) since the total cell numbers of thick-layer cultures are higher than 2D undifferentiated cultures used in (b). d. Soluble Aβ levels of the various enriched FAD ReN cells. The FAD cells with PSEN1ΔE9 showed elevated Aβ42/40 ratio as compared to the enriched ReN-GA cells.