Multidrop combi reagent dispenser

Each of the 1,948 compounds of the MIPE v4 library was tested in an 11-point dose-response starting at 46 μM with serial 1 to 3 dilutions. Briefly, cells were seeded into polystyrene, tissue-culture-treated 1,536-well white solid-bottom plates as previously reported (28 (link)). Seeding was performed with a Multidrop Combi Reagent dispenser (ThermoFisher) with a miniature 8-pin cassette at a density of 500 cells per well in 5 μL final volume. Immediately after dispensing the cells, 23 nL of compound stocks were transferred to individual wells using a Kalypsys pintool. The plates were then covered with stainless steel Kalypsys lids and incubated at 37°C with 5% CO₂ and 95% relative humidity for two days. 48 h post compound addition, 3 μL CellTiter-Glo reagent assay (Promega) was added to each well using a Multidrop Combi Reagent dispenser (ThermoFisher). Plates were incubated for 15 min at room temperature with the stainless-steel lid in place, and relative luminescence units (RLU) were quantified using a ViewLux imager (PerkinElmer) with a 2’ exposure time. Relative luminescence units for each well were normalized to the median RLUs from the DMSO control wells as 100% viability. The half-maximal activity concentration (LAC₅₀) values were calculated automatically from the fitting curves.

HSR (0.4 mg/ml) membranes were pre-incubated with 60 nM D-FKBP, for 90 min, at 37ºC, in a solution containing 150 mM KCl, 5 mM GSH, 0.1 mg/mL BSA, 1μg/mL Aprotinin/Leupeptin, 1 mM DTT and 20 mM PIPES (pH 7.0). To remove unbound D-FKBP, the SR membranes were spun down at 110,000 × g for 20 min, and then resuspended to 1 mg/mL in binding buffer consisting of 150 mM KCl, 5 mM GSSG, 0.1 mg/mL BSA, 1μg/mL Aprotinin/Leupeptin and 20 mM PIPES, pH 7.0. These samples were then incubated with 0.3 μM A-CaM for 30 min at 22ºC in binding buffer containing 0.065 mM CaCl₂ to give 30 nM Ca²⁺ in the presence of 1 mM EGTA (calculated by MaxChelator). This labeled SR sample was applied to the NCC assay plates in 50 μL aliquots using a Multidrop™ Combi reagent dispenser (Thermo Scientific) with a standard-tube cassette that loaded a single plate within 30 sec. An additional set of the NCC assay plates were loaded with HSR labeled with only D-FKBP, not A-CaM.

RPE-1 or MCF 10A-H2B-mCherry cells were plated in 384-well plates using the Multidrop Combi Reagent Dispenser (Thermo Scientific) at 250 and 500 cells per well respectively. To modulate the growth rate in RPE-1 cells, we induced expression the BRAF(V600E) oncogene by treating with indicated doses of doxycycline using a D300 Digital Dispenser (Hewlett-Packard). To modulate the growth rate in MCF 10A-H2B-mCherry we serum-starved cells twice with DMEM/F12 media supplemented with 0.1% bovine serum albumin and 1% penicillin-streptomycin. Media changes and cell washing was performed using a EL406 Microplate Washer Dispenser (BioTek). Cells were treated with indicated doses of human epidermal growth factor (EGF, eprotech) using a D300 Digital Dispenser. After 24 hours the cells were treated with a dilution series of etoposide using a D300 Digital Dispenser. RPE-1 cells were stained and fixed for analysis at the time of drug treatment and after 72 hours. MCF 10A-H2B-mCherry were imaged in an IncuCyte ZOOM live cell imager (Essen Bioscience) starting at the time of EGF treatment and drug sensitivity was evaluated 72 hours after drug addition.

Biochemical assays for WT EGFR (5 nM) and each mutant (L858R, 0.1 nM; L858R/T790M and L858R/T790M/C797S 0.02 nM) were carried out using a homogeneous time-resolved fluorescence (HTRF) KinEASE-TK (Cisbio) assay as described previously.^{20 (link)} Assays were optimized for each [ATP] = 100 μM final concentration. Inhibitor compounds in DMSO were dispensed directly in 384-well plates with the D300 digital dispenser (Hewlett Packard) followed immediately by the addition of aqueous buffered solutions using the Multidrop Combi Reagent Dispenser (Thermo Fischer). Compound IC₅₀ values were determined by 11-point inhibition curves (from 1.0 to 0.00130 μM) in triplicate. The data was graphically displayed using GraphPad Prism version 8.0 (GraphPad software). The curves were fitted using a non-linear regression model with a sigmoidal dose response.