Safire plate reader

HeLa or D17 cells were seeded in 24-well plates (Corning) at densities of 2.5x10⁴ cells/well and 2x10⁴ cells/well, respectively. Cells were infected 24 h later, with WT and mutant VLPs normalised on their RT activity and incubated at 37°C for 72 h. Cells were then lysed in Tropix Lysis buffer (Thermo Fisher Scientific) and frozen at -20°C. To measure LacZ activity, cell lysate was mixed with Tropix galactostar reaction mixture (Thermo Fisher Scientific) and luminescence was measured for 1 h at 10 min intervals on a Tecan Safire plate reader.
Absolute infectious titres of VLPs for microscopy assays were determined by X-gal staining of infected HeLa cells. HeLa cells were seeded in 12-well plates (Corning) at a density of 5x10⁴ cells/well and infected 24 h later with a 10-fold dilution series of VLPs by spinoculation (1600 g, 2 h, 16°C). Cells were then incubated at 37°C for 30 min prior to replacement of media with warm serum-supplemented DMEM. After 72 h, cells were washed in PBS and fixed for 10 min with 2% formaldehyde and 0.2% glutaraldehyde. Staining was performed overnight at 37°C in PBS with 0.4 mg/ml X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (Sigma), 4 mM K3Fe(CN)6 (Sigma), 4mM K4Fe(CN)6ˑ3H2O (Sigma), 2mM MgCl₂ (Thermo). The number of blue LacZ-expressing colonies were counted using a light microscope (Olympus) and the viral titre calculated.

Brains were homogenized in RIPA buffer using a sonicator (SONICS, USA) and serially diluted with ELISA sample buffer. Protein concentrations were determined using a BCA kit (Thermo Fisher Scientific). Quantitative measurements of Aβ1–40 and Aβ1–42 were performed using the Aβ1–40 and Aβ1–42 ELISA kits, respectively (IBL Japan). For each well, optical density at 450nm was measured on a Safire plate reader (Tecan, Switzerland), and Aβ1–40 and Aβ1–42 concentrations were determined by comparison with the corresponding standard curves. Total tau level was analyzed using total tau ELISA kit (Thermo Fisher Scientific) followed by user manual. The experiment was repeated three independent times.

10 μl of freshly collected plasma was diluted with 10 μl of water and added to individual wells of a 96-well plate preloaded with lyophilized TET reagent mixtures for negative, test and positive control wells (in triplicates). The readout luminescence signal was integrated for 0.4 seconds, and read continuously for 25 minutes using a TECAN Safire plate reader. For calculation of NSE levels, the linear regression slope for the initial activity was calculated per each well. Then, control wells (-2PG) were subtracted from the test channel and normalized to the positive control well (with 2-PG and enolase). Positive control reactions allowed us to normalize the results to reduce batch to batch variability. Plasma samples were sent for further analysis to ARUP laboratories (Salt Lake City, UT). Variability for PK and Luc activity between batches was addressed by preparing mixtures of the enzymes with equal activity rather than adding equal amounts of each protein.

Cells were washed in PBS and incubated for 15 minutes at 4°C and in NADOC (0.5% Tritonx-100, 0.5% sodium deoxycholate, 50 mM Tris, and 150 mM NaCl) lysis buffer (Thermo Fisher Scientific) with protease (HaltTM Protease Inhibitor Cocktail 100X, Thermo Fisher Scientific) and phosphatase inhibitors (HaltTM Phosphatase Inhibitor Cocktail 100X, Thermo Fisher Scientific). The extracts were centrifuged at 14,000g for 15 minutes. Protein concentrations in the supernatant were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific) and a Tecan Safire® plate reader. Protein extracts (25 μg) were resolved by 4-12% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (iBlot, Invitrogen). Membranes were blocked with SEABLOCK blocking buffer (Thermo-Scientific) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer. After washing in PBS-Tween buffer, the membranes were incubated with secondary antibodies conjugated to IRDye fluorophores. The infrared signal of the membranes was detected using an Odyssey detection system (Li-Cor biosciences).

The human neuroblastoma cell line SH-SY5Y was obtained from the European Collection of Cell Cultures (Centre for Applied Microbiology and Research). SH-SY5Y cells were cultured according to a previous report [79 (link)] with minor modifications. The cells were grown in MEM and GlutaMax medium (Gibco) and supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 units/mL penicillin, 100 μg/mL streptomycin (Gibco), and 1% non-essential amino acid solution (Gibco). Cultures were maintained in an incubator at 37°C with a humidified atmosphere of 5% CO₂. Freshly purified TTR (15 μM) in MEM was sterile-filtrated and pre-incubated with TBBPA, tafamidis, or diflunisal (15 μM each) for one hour before being added to the SH-SY5Y cells. The protein-compound solutions were supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin (Gibco), 1% non-essential amino acid solution (Gibco), 2 mM L-glutamine (Gibco), 1% MEM vitamins solution (Gibco), and 1% MEM amino acids solution (Gibco) and incubated with the cells for 48 h at 37°C in a humidified atmosphere of 5% CO₂. Cytotoxicity was measured using a resazurin reduction test, and cell viability was detected by fluorescence measurement using a TECAN Safire plate reader with excitation at 535 nm and emission at 595 nm. All experiments were performed in triplicate.

M-huPMCs and P-huPMCs (P0) were detached from culture dishes using TrypLE enzyme (Thermo Fisher Scientific) and seeded at a density of 1 × 10⁴ cells/well in 96-well plates. Cells were cultured to 80% confluence in DMEM/10% FBS and then starved overnight with Basal Medium Eagle (Gibco) supplemented with 1% FBS, 1% penicillin-streptomycin (P/S) and 1% Insulin-Transferrin-Selenium (ITS, Thermo Fisher Scientific). M-huPMCs and P-huPMCs (P1) were then treated with 100 μM H₂O₂, 15 μM CBR-5884 and 100 μM H₂O₂ + 15 μM CBR-5884 for 6 hr, respectively. A medium only, no treatment group was included as a control. The LDH concentration in each group was assayed by LDH cytotoxicity assay kit (Pierce). In brief, 20 µl supernatant of the 96-well plate was transferred into a 386-well plate. 20 µl reaction mixture was added into each well of the plate. The mixture was incubated at room temperature for 30 min. 20 µl stop solution was added to cease the reaction. The absorbance was measured at 490 nm and 680 nm with the plate reader (Safire², Tecan). To assess cell viability, 15 µl alamarBlue reagent was added to 150 µl medium in each well of the 96-well plate. After a 2 hr incubation at 37°C, the absorbance was read at 570 and 600 nm with the Tecan Safire plate reader (TECAN).