384 well plate

Antigen-specific antibody subclasses and isotypes, as well as FcγR binding, were analyzed by Luminex multiplexing in technical replicates. The antigens were coupled to magnetic Luminex beads (Luminex Corp) by carbodiimide- N-hydroxysulfosuccinimide (Sulfo-NHS) ester-coupling with an individual region per antigen. Coupled beads were incubated with different plasma dilutions (1:100 for IgG2, IgG3, IgG4, IgM, and IgA1; 1:500 for IgG1 and 1:1,000 for FcγR probing) for 2 hours at room temperature in 384 well plates (Greiner Bio-One). Unbound antibodies were removed by washing and subclasses and isotypes were detected with a respective phycoerythrin (PE)-conjugated antibody (anti-human IgG1 (Cat# 9052-09, RRID:AB_2796621), IgG2 (Cat# 9060-09, RRID:AB_2796635), IgG3 (Cat# 9210-09, RRID:AB_2796701), IgG4 (Cat# 9200-09, RRID:AB_2796693), IgM (Cat# 9020-09, RRID:AB_2796577), or IgA1 (Cat# 9130-09, RRID:AB_2796656) all from Southern Biotech) at a 1:100 dilution. For analysis of FcγR binding PE-Streptavidin (Agilent Technologies) was coupled to recombinant and biotinylated human FcγR2a, FcγR2b, FcγR3a, or FcγR3b protein. Coupled FcγRs were used as a secondary probe at a 1:1000 dilution. After a 1 hour incubation, excessive secondary reagent was removed by washing and the relative antibody concentration per antigen was determined on an IQue analyzer (IntelliCyt).

High-Throughput Screening was performed in 384-well plates (Greiner). First, compound was added to the plates using the Echo 520 (Labcyte). Next, HeLa-ActinLC3dNGLUC cells (20,000/well) were dispensed onto the compounds using the Thermo Fisher Multidrop and cultured for 24 h at 37°C. Supernatants (5 μl) were harvested and dispensed into black 384-well plates. Native coelenterazine (Cambridge Bioscience, #BT10110) in GLUC buffer (0.1% disodium phosphate, 5% glycerol, 150 mM sodium bromide, 1 mM EDTA, 25 mM Tris-HCL pH8 and 2 mM ascorbic acid) at a final concentration of 10 μg/ml was injected immediately prior to analysis using the Envision II (PerkinElmer) plate reader. For Z′ factor calculation, the following formula was used:
with STD_pos = standard deviation of the positive control and STD_neg = standard deviation of the negative control. For cell-based assays, we accept values that are higher than 0.3 for the Z′ factor.

Previously, we developed monoclonal HEK293T cell lines expressing constructs encoding human WT Aβ42 fused with YFP at the N terminus (26 ). Cell lines expressing human 4R tau (repeat domain) with the mutations P301L and V337M fused with YFP at the C terminus were generated as described previously (37 (link)).
To perform the bioassay, 3,000 cells per well (containing 0.1 μg/mL Hoechst 33342) were plated at 70 μL/well onto 384-well plates (Greiner) and incubated for 2 h before treatment with samples. Based on prior work (26 ), brain extracts (0.03×; 20% final volume) were incubated with Lipofectamine 2000 (1.5% final volume; Thermo Fisher Scientific) and Opti-MEM (78.5% final volume; Thermo Fisher Scientific) for 2 h. Following incubation, samples were plated onto 384-well plates in four replicate wells (10 μL/well). Plates were incubated, and DAPI and FITC channels were imaged every 24 h (five images per well) for 3 d using the GE Healthcare IN Cell Analyzer 6000. Images were analyzed using IN Cell Developer software and custom protocols containing algorithms to detect intracellular aggregates in live cells. Data are presented as integrated total fluorescence intensity per cell.

All inhibitors were provided by the drug discovery group at the Ontario Institute for Cancer Research (Toronto, ON, Canada). Cells were seeded at 1000–1500 cells/well into 384-well plates (Greiner Bio-One, Mississauga, ON, Canada). After 24 h, resistant cells were exposed to epirubicin at the selection doses established (see Flow cytometry section above), then exposed to histone deacetylase (HDAC) inhibitors (HDACi) dissolved in DMSO in 12 concentrations ranging from 0.0026 to 10 μM using HP D300 digital compound dispenser (Tecan Systems, San Jose, CA, USA). The DMSO concentration did not exceed 0.5 % in the final drug solution. After 72 h, the effects of inhibitors were determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) and the Wallac EnVision 2104 Multilabel Reader (PerkinElmer, Woodbridge, ON, Canada). Raw data were normalised to negative (media) and positive (20 μM staurosporine) controls and analysed using GraphPad Prism 5.