Alzheimer'sdisease and type 2 diabetes: Synthetic peptides may suppress formation of harmful amyloid aggregates TU_Muenchen NatureComms
; the final PK concentration in the assay was 0.5 µg/ml. fIAPP or hf-IAPP/ACM were prepared by incubating the peptides in 10 mM sodium phosphate buffer, pH 7.4 for 7 days ; fibril formation was confirmed by ThT binding and TEM. fAβ42 and hf-Aβ42/ACM were made as described under “ThT binding assays” . For IAPP-related assays, solutions were made by mixing 60 µl of the fIAPP or hf-IAPP/ACM solutions with 0.3 µl of the PK stock solution.
; mouse anti-Aβ for Aβ42); rabbit anti-Aβ40 for ACMs. Primary antibodies were combined with goat anti-mouse-POD or donkey anti-rabbit-POD ; detection was as under “Cross-linking, NuPAGE and WB”.
. Briefly, BV2 cells were obtained from Dr. M. Kipp ) and were maintained in GlutaMAX-supplemented RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin on poly--ornithine-coated flasks. For the phagocytosis assay, BV2 cells were seeded into 24-well plates containing coverslips in serum-free RPMI1640-GlutaMAX and further incubated for 24 h to reach 10,000 cells/well. Primary BMDMs were obtained from bone-marrow monocytes isolated from wildtype C57BL/6 mice ).
Following peptide incubation with the cells, supernatants were removed and cells on the coverslips were washed five times with ice-cold 1xPBS, fixed with 4% paraformaldehyde, washed with 1xPBS, permeabilized with 0.2% Triton-X 100, and rinsed three times with cold 1xPBS. Coverslips were mounted with Vectashield Antifade mounting medium containing DAPI . Images were acquired using a Leica DMi8 fluorescence microscope.
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