Presence of cell-associated and secreted oligomeric Aβ assemblies in primary neurons from the R1.40 transgenic mouse model of AD. A. Western blot analysis of detergent soluble cell extracts of from cultured primary cortical neurons (21 DIV) from non-transgenic (WT) and homozygous R1.40 (R/R) mice revealed the presence of numerous bands migrating between 14 and 38 kDa (arrows) that were immunoreactive with antibody 6E10 (recognizing residues 1–17 of Aβ1–42 peptide) in the R/R samples but not WT samples. Each lane for WT and R/R cultures represents extracts from three independent cultures. hAPP = holo APP; βCTF = β C-terminal fragment. Asterisk denotes the presence of non-specific bands detected with the human-specific 6E10 antibody. B. Primary cortical neurons (21 DIV) from WT and R/R mice were prepared and grown for 24 h in the presence of fresh NB media with (+) or without (-) the B27 serum supplement. Immunoprecipitatation of conditioned media with antibody 6E10, followed by Western blot analysis with the anti-Aβ oligomer specific antibody, NU-2 revealed the presence of multiple NU-2 reactive bands between 30–40 kDa (arrows) that were present in the R/R (lanes 5 and 6), but not WT (lanes 2 and 3) samples. Lanes 1 and 4 shows total input lysates. C-D. Aβ oligomers are secreted by R/R neurons. Cultured primary cortical neurons (21 DIV) from wild-type (WT) and homozygous R1.40 (R/R) embryos were double labeled with MAP2 (green in C and D) or Aβ oligomer specific antibodies NU-1 (red in C) or NU-2 (red in D). Merged images are shown in right panel. NU-1 and NU-2 immunreactivities were specifically observed in R/R, but not WT, neurons. Scale bar 30 μm.