Multidrop combi dispenser

For each cell line tested, a total of 2000 cells per well in 4 µL
of media were dispensed using a Multidrop Combi dispenser (Thermo Fisher
Scientific) and a small cassette into barcoded 1536-well flat-bottom white
(Corning (Corning, NY, USA)) collagen-coated plates. After a 24 h
incubation, library compounds and controls were added to assay plates at a
volume of 23 nL/well via a NX-TR pintool station (Wako Scientific Solutions (San
Diego, CA, USA)), and the plates were incubated further (another 24 h).
Firefly and Renilla luminescence outputs were measured sequentially using the
Dual-Glo Luciferase Assay System (Promega) and the ViewLux plate reader
(PerkinElmer). The assay’s performance was stable throughout the screen.
The activity of each compound was normalized to control wells (DMSO alone),
which were included on each plate. Cells treated with DMSO alone were defined as
having 0% activity. Of note, the following libraries were screened: the
library of pharmacologically active compounds (LOPAC),^{13 (link)} MIPE^{14 (link)} and the
NIH Chemical Genomics Center (NCGC) Pharmaceutical Collection
(NPC).¹⁵

Ldt_Mt2 (10 μM final concentration) was incubated with the inhibitor (100 μM final concentration) in the assay buffer (50 mM HEPES, pH 7.2, 0.01% (v/v) Triton X-100) for 2 hours at room temperature, then serially diluted 1000× with assay buffer (final Ldt_Mt2 concentration 10 nM). 20 μL of the diluted mixture was added to a black polystyrene, flat-bottomed 384-well μ-clear plate (clear bottomed, Greiner Bio-One, part number 781096). Probe 2 (final concentration 15 μM) was added using a MultiDrop Combi dispenser (ThermoFisher Scientific). Fluorescence was measured using a PHERAstar plate reader (BMG Labtech) with λ_ex = 480 nm and λ_em = 520 nm. Readings were taken every 15 s for a period of 5 minutes, using bottom optic measurements. The off rate (k_off) was determined using eqn (7). where, P = formed product; V_s = velocity of no-inhibitor control; V₀ = velocity of no-enzyme control, and P₀ = initial fluorescence.
The half-life of the enzyme–inhibitor complex (t_1/2) was calculated using eqn (8).

Neuro 2A cells were seeded (4,000 cells/well) in 4 µL of DMEM with 0.5% FBS and 1x Penicillin/Streptomycin in white solid bottom tissue-culture treated 1536-well plates (Greiner, 789173-F) with MultiDrop Combi Dispenser (Thermo Scientific). The cells were incubated for 24 h at 37 °C, 5% CO₂, 95% humidity. Twenty-three nL of tested compounds were transferred to each well with an automated pin-tool station (Kalypsys). Following incubation for 15 min at 37 °C, 0.5 µL IGF (500 ng/mL of final concentration) was added and incubated for additional 40 min at 37 °C. After the addition of 1.5 µL 4x lysis buffer and 15-min incubation at room temperature (25 °C), 2 µL of HTRF^® conjugated antibodies (fluorophore d2 conjugated anti-Akt monoclonal antibody and Europium³⁺ cryptate-labeled anti-pS473-Akt monoclonal antibody, obtained from CisBio, 1:80 dilution) were added with BioRAPTR FRD™ (Beckman Coulter). The plates were incubated at room temperature for 20 h. The signal was measured by EnVision Multilabel Plate Reader (PerkinElmer) with excitation at 330 nm, emission at 620 nm (donor) and 665 nm (acceptor). Results were calculated and analyzed in a format of the ratio 665 nm/620 nm multiplied by 10⁴.
Relative activity was calculated by normalizing each HTRF signal from each sample well to the mean HTRF signal from the DMSO-only control wells.