Spectramax plus 384

Reagents of analytical grade were used, and solvents were dried according to standard methods [26 ]. TLC was performed to monitor chemical reactions using aluminum plates coated with silica gel 60 F₂₅₄ from Merck (Darmstadt, Germany). Column chromatographic separations were performed using silica gel 60A 70–200 µ from Carlo Erba Reagents. Melting points (M.P.) were measured using a Leica Galen III hot-stage microscope. The NMR spectra (¹H and ¹³C) were recorded on a Bruker AVANCE III-400 NMR spectrometer (at 400 and 100 MHz). Tetramethylsilane (TMS) was used as a standard internal reference to report the chemical shifts (δ). Abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet. Mass spectra (ESI-MS) were obtained on a 500 MS LC Ion Trap mass spectrometer (Varian Inc., Palo Alto, CA, USA) equipped with an ESI ion source, operated in the positive ion mode. High-resolution mass spectra (HRMS) were obtained on a Bruker Impact II quadrupole mass spectrometer (Bruker Daltoniks, Billerica, MA, USA). UV-Vis spectrophotometers were used: Perkin Elmer Lambda 35 (radical scavenging, cholinesterase inhibition) and Spectramax Plus 384 (cell assays). Fluorescence measurements were performed on a microplate reader, BMG Labtech, POLARstar OPTIMA, for evaluation of Aβ₄₂ aggregation.

K2 cells were used as target cells for CDC assays. A total of 50 μL of cells was added to wells of a 96-well plates for a final concentration of 8 × 10⁴ cells per well in IMDM with Glutamax, 10% (w/v) heat-inactivated PBS, 1× NEAA, 1× sodium pyruvate, 1x penicillin-streptomycin. An additional 50 μL was added to the wells with or without test Abs and plates were incubated at 37 °C for 2 h. A total of 50 μL of 10% (w/v) rabbit complement (Invitrogen, Carlsbad, CA, USA) was added to the wells, and plates were incubated for 20 min at 37 °C. All samples were performed in triplicate. The plates were centrifuged at 200× g for 3 min, 50 μL of supernatant was removed to separate plates, and CDC was measured with a LDH cytotoxicity detection kit (Roche, Indianapolis, IN, USA). Absorbance was measured using a Spectra Max Plus 384 (PerkinElmer). Data were fit to a sigmoidal dose-response model using GraphPad Prism v6.0 software. Maximal cytotoxicity was obtained with Triton X-100 (Sigma-Aldrich) and spontaneous release with cells and complement alone. Specific cell lysis was calculated as follows: Cytotoxicity (%) = 100 × (optical density (OD) of sample − OD of spontaneous release)/(OD of maximal lysis − OD of spontaneous release).