Canine leiomyosarcoma low-passage cell line was cultured in DMEM + 10% FBS + 1% Penicillin/Streptomycin. Automated systems were used for a 119- and 2,100- compound high-throughput drug screens. The 119-drug screen library (Approved Oncology Set VI) was provided by the NCI Developmental Therapeutics Program (https://dtp.cancer.gov/ ). Automated liquid handling was provided by the Echo Acoustic Dispenser (Labcyte) for drug addition or Well mate (Thermo Fisher) for cell plating, and asays were performed using a Clarioscan plate reader (BMG Labtech). The BioActive compound library includes 2,100 small molecules that are annotated for pathway and drug target (Selleckchem) and was screened in triplicate. Compounds were stamped into 384 well plates for a final concentration of 1 μM using an Echo Acoustic Dispenser (Labcyte). Cells were then plated at a density of 2,000 cells/well using a WellMate (ThermoFisher) and incubated in the presence of drug for 72 h. After 72 h of incubation, Cell Titer Glo was added to each well and luminescence was measured using a Clariostar Plate Reader (BMG Labtech). Percent killing was quantified using the formula 100*[1-(average CellTiterGlodrug/average CellTiterGloDMSO)] where the value average CellTiterGloDMSO was the average DMSO CellTiterGlo value across each plate.
Echo acoustic dispenser
Manufactured by Beckman Coulter
Sourced in United States
The Echo acoustic dispenser is a laboratory equipment designed for non-contact liquid handling. It utilizes acoustic energy to precisely transfer small volumes of liquids without the need for physical contact, enabling efficient and contamination-free sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Wellmate, by Thermo Fisher Scientific (6 mentions)
Clariostar plate reader, by BMG Labtech (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Celltiter glo, by Promega (2 mentions)
Pherastar, by BMG Labtech (2 mentions)
Clarioscan, by BMG Labtech (2 mentions)
Prism 7, by GraphPad (1 mentions)
17 dmag alvespimycin hcl, by Selleck Chemicals (1 mentions)
Multidrop combi reagent dispenser, by Thermo Fisher Scientific (1 mentions)
Envision, by PerkinElmer (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Multiflo automated dispenser, by Agilent Technologies (1 mentions)
Camp gs dynamic kit, by PerkinElmer (1 mentions)
Multiflo, by Agilent Technologies (1 mentions)
Steady glo luciferase reagent, by Promega (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Transit 293, by Mirus Bio (1 mentions)
Pherastar fs, by BMG Labtech (1 mentions)
Mantis liquid dispenser, by Formulatrix (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
25 protocols using echo acoustic dispenser
1
Canine Leiomyosarcoma High-Throughput Drug Screening
2
Quantifying RORγ Inhibitor Potency
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As an alternate assessment of inhibitor potency MRL-871 was covalently labelled with Alexa Fluor-647. To determine the intrinsic affinity of the probe, the compound (5 mM DMSO stock) was serially diluted in threefold steps using an Agilent Bravo liquid handler. Diluted probe or DMSO (25 nl) were transferred into a black Greiner 384-well plate (Cat#781076) using a LabCyte Echo acoustic dispenser. To each well of the plate was added 15 μl of RORγ-LBD at 1.6, 3.33 and 6.65 nM in receptor buffer or receptor buffer alone. The plate was then incubated for 15 min at room temperature. Subsequently, 5 μl of receptor buffer with 1.2 nM Anti-His W1024 Europium chelate antibody (PerkinElmer) was added and the TR-FRET signal measured as in the SRC-1 cofactor recruitment assay described above. TR-FRET intensity in the presence of RORγt was corrected for background and the KD determined using the One-site specific binding equation in Graph Pad Prism. To assess the potency of unlabelled compounds, a similar assay format was utilized except that 100 nM probe was preincubated with the RORγt-LBD for 15 min before addition to serially diluted compounds. IC50 values were determined by fitting per cent activity data to a four-parameter logistic dose–response equation in GraphPad Prism.
3
Fluorometric OTUB2 Inhibition Assay
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The assays were performed in
“non-binding surface flat bottom low flange” black 384-well
plates (Corning) at room temperature in a buffer containing 50 mM
Tris·HCl, 100 mM NaCl, pH 7.6, 2.0 mM cysteine, 1 mg/mL 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic
acid (CHAPS), and 0.5 mg/mL γ-globulins from bovine blood (BGG).
Each well had a final volume of 20.4 μL. The compounds were
dissolved in 10 mM DMSO stocks, and appropriate volumes were transferred
to the empty plates using a Labcyte Echo acoustic dispenser. A DMSO
back-fill was performed to obtain equal volumes of DMSO (400 μL)
in each well. N-Ethylmaleimide (NEM, 10 mM) was used
a positive control (100% inhibition) and DMSO as negative control
(0% inhibition). A 10 μL portion of buffer was added, and the
plate was vigorously shaken for 20 s. Next, 5 μL of OTUB2 (full
length) was added to a final concentration of 25 nM followed by incubation
for 30 or 150 min. A 5 μL portion of the substrate (Ub-Rho)
was added (final concentration 400 nM), and the increase in fluorescence
over time was recorded using a BMG Labtech Clariostar plate reader
(excitation 487 nm, emission 535 nm). The initial enzyme velocities
were calculated from the slopes, normalized to the positive and negative
controls, and plotted using GraphPad Prism 7 to obtain the IC50 values.
“non-binding surface flat bottom low flange” black 384-well
plates (Corning) at room temperature in a buffer containing 50 mM
Tris·HCl, 100 mM NaCl, pH 7.6, 2.0 mM cysteine, 1 mg/mL 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic
acid (CHAPS), and 0.5 mg/mL γ-globulins from bovine blood (BGG).
Each well had a final volume of 20.4 μL. The compounds were
dissolved in 10 mM DMSO stocks, and appropriate volumes were transferred
to the empty plates using a Labcyte Echo acoustic dispenser. A DMSO
back-fill was performed to obtain equal volumes of DMSO (400 μL)
in each well. N-Ethylmaleimide (NEM, 10 mM) was used
a positive control (100% inhibition) and DMSO as negative control
(0% inhibition). A 10 μL portion of buffer was added, and the
plate was vigorously shaken for 20 s. Next, 5 μL of OTUB2 (full
length) was added to a final concentration of 25 nM followed by incubation
for 30 or 150 min. A 5 μL portion of the substrate (Ub-Rho)
was added (final concentration 400 nM), and the increase in fluorescence
over time was recorded using a BMG Labtech Clariostar plate reader
(excitation 487 nm, emission 535 nm). The initial enzyme velocities
were calculated from the slopes, normalized to the positive and negative
controls, and plotted using GraphPad Prism 7 to obtain the IC50 values.
4
High-Throughput Screening of Osteosarcoma Cell Lines
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All human (143B, MG63, SAOS, U2OS, 17-3X) and canine (Abrams, Moresco, D17, D418) osteosarcoma cell lines were cultured in DMEM + 10% FBS + 1% Penicillin/Streptomycin. The NIH Approved Oncology set (119 compounds) and Selleck Bioactives collection (2100 compounds) were screened in the Duke Functional Genomics Shared Resource as described previously [21 (link),50 (link)]. Briefly, compounds were first stamped in triplicate into 384 well plates at a final concentration of 1 μM using an Echo Acoustic Dispenser (Labcyte, Indianapolis, IN, USA). Cells and media were then dispensed into plates using a WellMate (Thermo Fisher, Waltham, MA, USA) at a density of 2000 cells/well for each cell line. CellTiter-Glo (Promega, Madison, WI, USA) viability assays were performed after incubation of cells with compounds for 72 h and luminescence was read using a Clariostar plate reader (BMG, Berlin, Germany). Top drug targets as identified by the high-throughput drug screens, Bortezomib (PS-341), Verdinexor (KPT-335) and 17-DMAG (Alvespimycin) HCl were purchased from Selleck Chemicals (Houston, TX, USA) and were solubilized in DMSO at 10 mM concentration to use for in vitro IC50 studies. Dose-response curves were generated with four-fold serial dilutions from a starting concentration of 20 μM.
5
High-Throughput Screening of Anti-Giardia Compounds
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Each compound was assessed in singlicate, at a final concentration of 10 μM (0.4 μL of a 5 mM stock; diluted to a final volume of 200 μL in culture medium; 0.2% DMSO). All compounds and controls (0.4 μL DMSO and 0.4 μL 5 mM albendazole; final 10 μM) were plated by Compounds Australia using an Echo acoustic dispenser (Labcyte, Sunnyvale, CA, USA) before the addition of media inoculated with BRIS/91/HEPU/1279 trophozoites (200 μL final volume; 1.5 × 104 trophozoites per well). Plates were incubated in sealed chambers filled with 3% O2, 5% CO2 in N2 and growth was assessed at 24 and 48 h using live cell imaging and automated counting as previously described [23 (link)]. Growth inhibition was calculated by subtracting media only background counts and determining percentage growth relative to vehicle (0.2% DMSO) controls. The reliability of these assays was assessed by calculating Z’ factors for each plate as previously described [24 (link)]. Growth inhibitory activity was reassessed (in singlicate) if compounds demonstrated ≥50% growth inhibition or if they belonged to a scaffold of interest.
6
Dose-response screen of BDCS library
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The library used in this screen is an internal GSK collection of 5857 biological target-annotated compounds (Biologically Diverse Compound Set, BDCS). The compounds were transferred to 384-well Greiner plate at 300nl/well using Echo acoustic dispenser (Labcyte). Column 6 (all 16 wells) contained DMSO only (low control, 0% inhibition). Column 18 (16 wells) contained compound II at 1mM concentration in DMSO (high control). 30ul of cells in the culture media was dispensed onto the compound plate (1:100 dilution of the compounds, 1% DMSO final concentration). Compounds were tested as dose response starting at a stock concentration of 10mM and diluted serially 3-fold across 11 points, also in DMSO.
7
High-Throughput Screening of FDA Compounds
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We used a library of 1430 FDA approved compounds from various commercial suppliers with validated biological and pharmacological activities. For single concentration screening 5 nL of 10 mM compound solution in DMSO was transferred into HiBase Low Binding 384-well flat bottom plates (Greiner bio-one) to give a final screening concentration of 10 μM. Columns 23 and 24 were reserved for uninhibited and inhibited controls respectively. The uninhibited control contained 5 nL DMSO but no compound, whereas the inhibited control contained 5 nL PR-619 but the enzyme was inactivated by pre-treatment with 1.0 % TFA. All compounds and DMSO were dispensed using an Echo acoustic dispenser (Labcyte, Sunnyvale, USA). For all HTS assays the final DMSO concentration was 0.1 %. For concentration response curves of known HOIP, MDM2 and ITCH inhibitors, a threefold serial dilution was prepared from 10 mM compound solutions in DMSO in 384-well base plates V-Bottom (Labtech). 100 nL of compound was transferred into 384-well round bottom low binding plates using a Mosquito Nanoliter pipetter (TTP Labtech, Melbourn, UK), giving a final concentration range between 100 μM and 100 nM.
8
Cell Viability Screening Assay Protocol
9
Automated Screening of FDA-Approved Drugs
10
Automated Yeast Growth Assay
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125 nL of compounds or DMSO were dispensed into 384-well plates using the Echo acoustic dispenser (LabCyte) to achieve a final concentration of 25 µM. 50 µL of 10−2 dilution of an OD600 = 1.0 or 0.5 yeast cell suspensions were dispensed into the 384-well plates containing compounds or DMSO with a MultiFlo automated dispenser (Biotek). Plates were covered and incubated at 30° for 16 – 21 hr until OD600 reaches ∼0.8. Plates were vortexed briefly to resuspend cells prior to reading.
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