*5.15. Enzyme-Linked Immunosorbent Assay (ELISA)*

CHO-APP and SK-N-SH cells were seeded onto 24-well plates. The following day, cells were treated with 400 µL/well verapamil, nicardipine, DMSO or plain cell culture media. Because SK-N-SH cells have been reported to secrete low concentrations of Aβ peptides (in the picogram/mL range) [51,52,92,93], an experiment period of 24 h was selected to allow time to accumulate sufficient peptide in the cell supernatant for detection using ELISA. After 24 h, media from each well was collected, treated with 1 µL protease inhibitor cocktail (equivalent to 0.25 mM AEBSF), centrifuged at 3000× *g* RPM at 4 ◦C for 5 min to remove debris, and used immediately for ELISA (to avoid peptide degradation due to storage and freeze/thawing). Cellular secretion of Aβ<sup>40</sup> and Aβ<sup>42</sup> into media was detected and quantified by sandwich ELISA.

For CHO-APP supernatant, a pair of in-house isoform-specific ELISAs were developed: analyses were performed in 96-well Nunc-ImmunoTM MaxiSorp plates (Thermo Fisher Scientific) coated with 100 µL per well of capture antibody at 2.5 µg/mL in 0.1 M carbonate buffer (pH 9.5) at 4 ◦C overnight. Wells were washed four times with PBS-Tween (0.05% *v*/*v*) to remove any unbound antibody, before blocking with 200 µL of blocking buffer (2% BSA, 7.5 g/L glycine in PBS) for 1 h at room temperature. After another four washes, 100 µL peptide standards and samples were loaded into the wells and incubated for 1 h at room temperature on a slow-speed shaker. Washing was repeated, then wells were incubated with 100 µL of the appropriate primary detection antibody (1:1000 in 1% BSA, 3.75 g/L glycine in PBS) for 1 h at room temperature. After washing, 100 µL of secondary horseradish peroxidase-conjugated antibody (1:10,000 in 1% BSA, 3.75 g/L glycine in PBS) was added, and the plate was incubated for 1 h at room temperature. Following another four washes, 100 µL of TMB was added to each well. Plates were incubated in the

dark for 30 min at room temperature, then the reaction was terminated by the addition of 50 µL of H2SO<sup>4</sup> (20% *v*/*v*). Absorbance values at 450 nm were measured using a Bio-Rad Microplate Reader. Assay sensitivity was <300 pg/mL for both Aβ<sup>40</sup> and Aβ42.

For SK-N-SH supernatant, the commercial human Aβ<sup>40</sup> and Aβ<sup>42</sup> ELISA kits (Invitrogen, catalogue no. KHB3481 and KHB3544) were used in accordance with the manufacturer's protocols.

Data were computed using Graphpad Prism software; a non-linear 4-parameter regression was used to generate a standard curve, from which the unknown concentrations were determined. ELISA data were adjusted for total protein content of each sample (as determined by BCA protein assays) to account for any potential slight variations in cell viability, by multiplying the ELISA concentration by the ratio of control protein concentration to sample protein concentration.
