Additional file 3: of Accumulation of amyloid-β by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons

Intracellular localization of large Aβ42-555 inclusions in astrocytes. Intersections from a 3D z-stack of an astrocyte exposed to Aβ42-555 protofibrils demonstrate that Aβ42-555 deposits are located intracellularly (arrow heads). DAPI (blue), GFAP (green) and Aβ42-555 (red). Scale bars: 10 μm. (TIF 2239 kb

Additional file 13: of Accumulation of amyloid-β by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons

Aβ42 protofibril characterization. No considerable difference is noticed comparing serially diluted Aβ42 protofibrils and Aβ42-555 protofibrils with the Aβ protofibril selective mAb158 ELISA. (TIF 149 kb

Additional file 4: of The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

The irrelevant antibody MOPC-173 has a significantly lower effect on Aβ accumulation in astrocytes than mAb158. Aβ42 protofibrils were accumulated in astrocytes (A), and addition of mAb158 to the co-cultures lowered the accumulation of Aβ42 protofibrils (B). Addition of the irrelevant antibody MOPC-173 partly lowered the Aβ42 accumulation in astrocytes (C). The total 555-intensity was analyzed per number of live cells (D) and number of inclusions (E), and the total 555-area per number of inclusions (F). Taken together, the analysis shows that mAb158 had a significantly higher effect on the Aβ accumulation, compared to MOPC-173. Phalloidin (green), DAPI (blue), Aβ42 (red). Scale bar: 20 μm. The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed using Mann-Whitney U-test (**P < 0.01 and ***P < 0.001). (TIFF 5931 kb

Additional file 6: of The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

RmAb158 reduces Aβ inclusions in astrocytes. Co-cultures were exposed to Aβ42 protofibrils (A) or Aβ42 protofibrils together with RmAb158 (IgG2c) (B). Measurements of the total 555-intensity per number of live cells (C) and number of inclusions (D), and the total 555-area per number of inclusions (E) confirmed that RmAb158 reduces Aβ inclusions in astrocytes. GFAP (green), DAPI (blue), Aβ (red). Scale bar: 20 μm. The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed using Mann-Whitney U-test (***P < 0.001). (TIFF 4151 kb

Additional file 3: of The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

The mAb1C3 lowers Aβ inclusions in astrocytes. Aβ42 protofibrils were accumulated in astrocytes (A), but addition of the mAb1C3, binding pan-Aβ, to the co-cultures lowered the accumulation of Aβ42 protofibrils (B). The total 555-intensity was analyzed per number of live cells (C) and number of inclusions (D), and the total 555-stained area per number of inclusions (E). Taken together, the analyses confirmed that mAb1C3 lowers Aβ42 inclusions in astrocytes. Phalloidin (green), DAPI (blue), Aβ42 (red). Scale bar: 20 μm. The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed using Mann-Whitney U-test (***P < 0.001). (TIFF 6026 kb

Additional file 2: of The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

The irrelevant antibody Ly-128 does not reduce the intracellular Aβ accumulation. The Aβ aggregates that were formed in astrocytes in Aβ42 protofibril exposed cultures (A) were clearly reduced in the presence of mAb158 (B). To ensure that this effect was specific for Aβ antibodies, mAb158 was exchanged to the irrelevant antibody Ly-128 (IgG1) (C). Ly-128 did not reduce the intracellular Aβ accumulation. In addition, mAb158 had only a minor effect on the Aβ accumulation if it was added to the co-cultures for 1 h prior to the Aβ42 protofibril exposure (D). mAb158 had a significantly higher effect on the Aβ accumulation, compared to mAb158 (1 h). The total 555-intensity was analyzed per number of live cells (E) and number of inclusions (F), and the total 555-stained area per number of inclusions (G) significantly decreased when treated with mAb158 compared to Aβ42 protofibril or Aβ42 protofibril + Ly-128 exposed co-cultures. GFAP (green), DAPI (blue), Aβ42 (red). Scale bar: 20 μm. The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed using Mann-Whitney U-test (**P < 0.01 and ***P < 0.001). (TIFF 5989 kb

Additional file 5: of The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

Aβ42-555 protofibrils and mAb158 are engulfed and co-localize inside astrocytes. Time-lapse recording demonstrating co-localization between Aβ42-555 protofibrils and DyLight™ 488 labeled mAb158 antibody in astrocytes (A). However, in the presence of antibodies, Aβ42-555 protofibrils reached much weaker signals, compared to cultures exposed to Aβ42-555 protofibrils only (B). Scale bars: A and B = 10 μm. (TIFF 6980 kb

Additional file 7: of The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

The heavy and light chain of the mAb158 antibody can be detected in the media. Reprobing the filter in Fig. 5 with a secondary anti-mouse IgG antibody, showed that both the heavy and light chain of the mAb158 antibody can be detected in the media of antibody-treated cultures. (TIFF 2941 kb

Fractionation of transgenic mouse brain extracts.

<p>Fractions of mouse brain homogenate from AβPP_ArcSwe transgenic mice (n = 5) and non-transgenic littermates (n = 5) were analyzed with Aβ1–42 (A), Aβ1–40 (B) and Aβ protofibril specific (C) ELISAs. Horizontal lines indicate the mean value of each group. Immunoprecipitation of pooled material from fraction 2 of transgenic mouse brain homogenate with the conformation specific Aβ protofibril selective antibody mAb158 covalently coupled to Dynabeads (0 – non immunoprecipitated sample, IP – immunoprecipitated material, S – supernatant remaining after ip) (D). Error bars indicate the standard deviation.</p

Fractionation of human brain extracts.

<p>Fractions of human brain homogenate from temporal cortex of diseased AD patients (n = 7, including one AβPP_Swe and one AβPP_Arc mutation carrier) and non-AD subjects (n = 4, one control subject and three FTD patients) were analyzed with Aβx-42 (A), Aβx-40 (B), Aβ1–42 (C) and Aβ1–40 (D) ELISAs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032014#s2" target="_blank">Results</a> from the individual carrying the Swedish mutation is marked ‘Swe’ in the Aβx-40 and Aβ1–40 graphs. Horizontal lines indicate the mean value of each group. The level of N-terminal truncation of Aβ42 was determined as a ratio between Aβ1–42 and Aβx-42 (1-[Aβ1–42]/[Aβx-42]) (E), with error bars indicating the standard deviation. Immunoprecipitation of pooled material from fraction 2 of AD brain and non-AD brain with the conformation specific Aβ protofibril selective antibody mAb158 covalently coupled to Dynabeads (0 – non immonuprecipitated sample, IP – immunoprecipitated material, S – supernatant remaining after ip) (F). Error bars indicate the standard deviation.</p