The Vero CCL81 cells were seeded at a density of 1.7 × 103; cells per well in a flat clear bottom, 384-well assay plate (Greiner Bio-One, Kremsmünster, Austria), cultured in 45 μL of DMEM, supplemented with 10% FBS (v/v), and maintained at 37 °C and 5% CO2. After 24 h, 1 μL of the MMV COVID Box compound library at 10 mM was added to 40 μL of DMEM in each well of an intermediate plate and, then, 15 μL was transferred to the assay plates for a final concentration of 20 μM. Cells were infected in the presence of compounds at an MOI of 0.5, by adding 15 μL of virus diluted in FBS-free media, and incubated for 72 h. The cells were stained with Hoechst-33342 after the supernatant was discarded and fixed with 4% paraformaldehyde (PFA).
384 well assay plate
Manufactured by Greiner
Sourced in Austria, United States
The 384-well assay plates are a laboratory equipment designed for high-throughput screening and sample processing. They feature a grid of 384 individual wells, allowing for the simultaneous testing or analysis of multiple samples or compounds.
Automatically generated - may contain errors
Lab products found in correlation
Echo 550, by Beckman Coulter (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Probenecid, by Thermo Fisher Scientific (1 mentions)
Multidrop combi, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Dojindo Laboratories (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Alphascreen assay, by PerkinElmer (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Phenol red free mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mixmate, by Eppendorf (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
7 protocols using 384 well assay plate
1
SARS-CoV-2 Inhibitor Screening in Vero Cells
2
High-Throughput Anticancer Drug Screening
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The US Food and Drug Administration (FDA)-approved investigational drug library (41 compounds, 10 mM in DMSO; bortezomib as a positive control) and the drug library used for comparative response analysis (Supplemental Figure S3 and Supplemental Table S4 ) were purchased from Selleckchem Inc. (Houston, TX, USA), except for LNT1, which was purchased from Tocris. The compounds used in the screen on PD-GBOs are known anticancer molecular agents including inhibitors of epidermal growth factor (EGFR), fibroblast growth factor (FGFR), vascular endothelial growth factor (VEGFR), mechanistic target of rapamycin (mTOR), and phosphoinositide 3-kinase (PI3K). The compounds of the library were transferred to a 384-well assay plate (Greiner Bio-One) that was designed as a fourfold and seven-point serial dilution series (maximum 30 µM in 40 µL of culture medium) using the Echo 550 liquid handler (Labcyte, San Jose, CA, USA). Prepared drug assay plates were sealed and stored at −80 °C until further use. We used 80 different drugs, with each concentration tested twice for the comparative response analysis on printed U87 spheroids. A total of 13 drugs were used for both comparative response analysis and PD-GBO screening.
3
Fluorescent Calcium Imaging Assay
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Cultured cells were washed with PBS and trypsinized with 1 mL 0.05% trypsin-EDTA. To load the fluorescent probe into the cells, the PBS-washed cell pellet was resuspended in 2 mL of loading buffer with 1μM Fluo-4 AM (Dojindo), 0.04% Pluronic F-127 (Sigma-Aldrich), 2.5 mM Probenecid (Thermo Fisher Scientific), and 20 mM HEPES (Thermo Fisher Scientific) in HBSS (Thermo Fisher Scientific). The cell suspension was incubated for 30 min at 37°C under light shielding conditions. After centrifugation and removal of the supernatant, the precipitate was resuspended in 8 mL of recording buffer (2.5 mM Probenecid and 20 mM HEPES in HBSS). The cells were dispensed at 20 μL/well using a Multidrop Combi (Thermo Fisher Scientific) into a 384-well assay plate (Greiner) in which 100 nL of 1 mM compound solution was dispensed in advance. After 1 h of incubation at 37°C with light shielding, a calcium assay was performed.
4
Quantifying Tau Proteins and Phosphorylation
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AlphaScreen assays (Perkin Elmer) were performed according to the manufacturer’s guidelinesusing tau-specific antibodies. Optimised Alpha-Screen assays were performed using either biotinylated DA9 (bDA9) and acceptor (Ab-ACC) bead antibodies: TG5 total tau, CP13 pS202-tau, MC6 pSer235-tau, PHF1 pSer396/pSer404-tau, or anti-Tau 421, and 10μl per well of optimized antibody mix of Ab-ACC and biotinylated antibody were added to 384-well assay plates (Greiner) together with 5μl per well of sample or standard diluted in AlphaScreen assay buffer (0.1% casein in DPBS). Plates were incubated overnight in the dark at 4°C. After overnight incubation, 5μl of the streptavidin-coated donor beads diluted in AlphaScreen buffer were added to each well and plates incubated in the dark with gentle agitation at room temperature for 4 h. Plates were read at an excitation wavelength 680 nm and emission at 520–620 nm using an Envision plate reader (Perkin Elmer). Linear regression with variable slope analyses was used to calculate relative amounts of total tau and phospho-tau from standard curves derived using purified PHFs. Relative amounts of D421-caspase cleaved tau were calculated from the AlphaScreen count levels relative to the standard curves derived using N2a cells transiently expressing 2N4R tau and treated with 0.5μM staurosporine.
5
High-Throughput Screening of CTPilB Inhibitors
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The HTS was conducted at the Virginia Tech Center for Drug Discovery (VTCDD) Screening Laboratory using the Agilent Bravo automated liquid-handling platform with 384-well assay plates (Greiner; 781101). Each assay well contained CtPilB at 0.5 μM, MANT-ATP at 0.4 μM, and a library compound at 20 μM in 20-μl reaction mixtures in activity buffer. Fluorescence was measured using a SpectraMax M5 multimode microplate reader at room temperature. For the development and optimization of the HTS, ATP at 20 μM was used as the positive control because it was viewed as an inhibitor of the binding between MANT-ATP and CtPilB, whereas samples without ATP were the negative controls for the absence of an inhibitor. The Z′ factor for this assay was calculated using the following equation (40 (link)): Z′ = 1 − [3 × (SD+ + SD−)/(μ+ − μ−)], where SD is the standard deviation and μ is the fluorescence intensity. The plus and minus subscripts refer to MANT-ATP and MANT-ATP with CtPilB, respectively.
6
High-throughput HIV Infection Screening
7
Glycogen Synthase Complex Activity Assay
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The activity of GYS1–GYG1 complexes was measured using the UDP-Glo GT (Promega) according to the manufacturer’s instructions. To measure activity, 10 μl per well of each reaction containing 100 nM GYS1–GYG1, 1 mM UDP-glc, 0.5 mg ml−1 glycogen and 10 mM Glc6P in assay buffer (25 mM HEPES, pH 7.5, 200 mM NaCl, 0.5 mM TCEP) was dispensed into 384-well assay plates (Greiner). Following a 60-min incubation at room temperature, 10 μl of UDP-Glo Plus detection reagent was added (final assay volume: 20 μl per well) and, after a further 60 min of room-temperature incubation, luminescence was detected using a SpectraMax M3 (Molecular Devices).
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