Single-channel Ca(2+) imaging implicates Aβ1-42 amyloid pores in Alzheimer’s disease pathology.

Preparation and characterization of Aβ1–42 oligomers

Soluble oligomers were prepared by dissolving 0.5 mg of human recombinant Aβ1–42 peptide (hexafluoroisopropanol pretreated; rPeptide) in 20 µl of freshly prepared DMSO and were quickly diluted with 480 µl of double-distilled water in a siliconized Eppendorf tube. After a 10-min sonication, samples were incubated at room temperature for 10 min and then centrifuged for 15 min at 14,000 g . The supernatant fraction was transferred to a new siliconized tube and stirred at 500 rpm using a Teflon-coated microstir bar for 8–48 h at room temperature. Aliquots were taken at intervals and were assayed by pipette application to voltage-clamped oocytes at a final bath concentration of 1 µg/ml. Membrane currents were recorded 30 min after application in response to hyperpolarization from 0 to −100 mV. Aβ preparations incubated for 0.2, a threshold value empirically determined for our experimental conditions. Events that do not fulfil either of these criteria are assigned to new pore locations. Finally, putative pore locations from the initial list that are not associated with any events are discarded. By the end of the detection and attribution processes, a final list of designated pore locations has been produced, and each location is associated with a list of linked events. Data visualization, analysis, and export. The list of events associated with each pore location is used to derive distributions of pore open and closed times (i.e., event durations and inter-event intervals), from which population statistics are then calculated. A graphical user interface allows on-screen viewing of raw and processed data (e.g., intermediate stack, baseline, signal, and noise information). Clicking on a map of locations brings up plots as these values as functions of time, and sampling can be modified to select a mean or maximum of pixel values over a user-specified region of interest. Data (including individual measurements of event durations, intervals, and fluorescence amplitudes together with population measurements of mean pore open/closed times, P o , and mean fluorescence) are exported as ASCII text files. Materials Fluo-4 dextran was purchased from Invitrogen, and human Aβ1−42 peptide was purchased from Millipore and rPeptide. gsmtx-4 was a gift from F. Sachs (University at Buffalo, Buffalo, NY). All other reagents and antibodies were purchased from Sigma-Aldrich.

Materials Fluo-4 dextran was purchased from Invitrogen, and human Aβ1−42 peptide was purchased from Millipore and rPeptide. gsmtx-4 was a gift from F. Sachs (University at Buffalo, Buffalo, NY). All other reagents and antibodies were purchased from Sigma-Aldrich.