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Optiplate 384

Manufactured by PerkinElmer
Sourced in United States

The OptiPlate-384 is a 384-well microplate designed for high-throughput screening applications. It features a black well bottom and white well walls, optimized for luminescence and fluorescence detection. The plate is made of polystyrene and has dimensions of 128 mm x 86 mm.

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31 protocols using optiplate 384

1

AlphaScreen Assay for Bromodomain Inhibitors

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The AlphaScreen assay was described previously and performed according to the manufacturer’s protocol (PerkinElmer, USA). The reaction buffer was 50 mM HEPES pH 7.4, 100 mM NaCl, 0.1% BSA, and 0.05% CHAPS. The whole reaction and screening were performed using OptiPlateTM-384 (PerkinElmer, Waltham, MA, USA).
The compounds, acetylated peptide [SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-biotin] and purified bromodomain protein were added to the OptiPlateTM-384 plate. Then, streptavidin-coated donor beads and anti-GST AlphaScreening acceptor beads were added. The incubation condition was 25 °C for 1 h using a Thermomixer C (Eppendorf, USA). The blocking efficacy (IC50) values were obtained by 8-point titration (from 0.0096 to 32 μM). The IC50 values of inhibitors were evaluated with GraphPad Prism 7 using normalized and inhibition analysis suites.
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2

cGMP Quantification Assay Protocol

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A biotinylated cGMP supplement containing an anti-cGMP antibody, protein A-coated acceptor beads and streptavidin-coated donor beads was purchased from Perkin Elmer Corp. (Waltham, Massachusetts, USA). Biotinylated cGMP was prepared at a concentration of 5 nM in assay buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% Tween-20) containing 1 mg/mL bovine serum albumin (BSA). The acceptor bead mix was prepared in assay buffer with the anti-cGMP antibody (diluted 1:2000) and protein A-coated acceptor beads (50 μg/mL). streptavidin-coated donor beads were prepared in assay buffer at a concentration of 100 μg/mL. The cGMP standard was prepared as a serial dilution from 0.5 nM to 5 μM in assay buffer without BSA. Lyophilized samples were resuspended in assay buffer without BSA. The reaction was carried out in white opaque 384-well plates (Optiplate TM-384, Perkin Elmer) with 10 μL of the acceptor bead mix in each well together with 5 μL of cGMP standard dilutions or lyophilized samples. The plates were incubated for 20 min at room temperature in the dark before adding 5 μL of biotinylated cGMP and incubating for a further 5 min at room temperature. Finally, 5 μL of diluted streptavidin-coated donor beads was added and the reaction was incubated for 2 h at room temperature in the dark. Fluorescence was then measured using the Enspire multi-label plate reader (Perkin Elmer).
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3

Tau Aggregate Detection Assay

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Cells in 96-well plates were lysed in 40 μL/well RIPA buffer with protease and phosphatase inhibitors (Roche). After 20–30 min of gentle shaking at RT, a 5 μL sample was mixed with 20 μL of biotinylated and acceptor-bead-conjugated antibodies in OptiPlate-384 (all Perkin Elmer). After 2 hr of incubation at RT, 25 μL of streptavidin donor beads were added at RT for 30 min followed by detection with the Envision plate reader. Raw values were normalized to transduced control samples (without fibrils) per plate. To allow the detection of aggregates, the monoclonal hTAU10 antibody was conjugated to both acceptor beads and biotin. To measure total tau levels, a biotinylated HT7 antibody was combined with acceptor-bead-conjugated hTAU10 (HT7/hTAU10) (Verheyen et al., 2015 (link)).
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4

Sandwich Immunoassays for Tau Quantification

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A comprehensive panel of sandwich immunoassays listed in Table 1 was developed to measure different forms of tau. In general, analytes were captured by a monoclonal antibody that was biotinylated and bound to streptavidin coated donor beads (PerkinElmer). Detection was accomplished either by a monoclonal antibody conjugated to the acceptor beads directly or by a polyclonal antibody in combination with anti-rabbit IgG-conjugated acceptor beads (PerkinElmer).
Assay reactions (25 μl) were carried out in OptiPlate-384 microplates (PerkinElmer) that contained 5 μL of analyte at the specified protein concentration, 10 μL of biotinylated capture antibody (final concentration 2 nM) and 10 μL of detection antibody-conjugated acceptor beads (final concentration 20 μg/mL). After overnight incubation at 4°C, 25 μL of streptavidin donor beads were added under subdued light conditions (final concentration 40 μg/mL) and the reactions were incubated at room temperature for 60 min with gentle shaking. The fluorescent signal was detected on an Envision Plate Reader at 615 nm (PerkinElmer).
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5

Evaluating Peptide Binding to Influenza Hemagglutinin

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The AlphaScreen assay was used to assess the binding between recombinant HA and its receptor mimic, biotinylated sialyllactose polymer α2–3-sialyllactose-polyacrylamide (PAA) (Glycotech) as follows17 (link). Various concentrations of tetravalent peptides were incubated with HA (10 μg/ml) in individual wells of an OptiPlate-384 (PerkinElmer) for 30 min at room temperature. After the addition of α2–3-sialyllactose-PAA (16 nm), the plate was further incubated for 1.5 h. The samples were then incubated with nickel chelate acceptor beads (20 μg/ml; PerkinElmer) for 30 min and then with streptavidin donor beads (20 μg/ml; PerkinElmer) for 1 h at room temperature in the dark. The plate was then subjected to excitation at 680 nm, and emission from the wells was monitored at 615 nm with an EnVision system (PerkinElmer). Data were obtained as the AU of signal intensity (count per second) used by the EnVision system.
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6

Quantitative Protein Analysis via AlphaLISA

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Cells in 96w plate were lysed in 40μ/well RIPA buffer with protease-and phosphatase inhibitors (Roche). After 20–30 minutes of gentle shaking at RT, 5μl sample was mixed with 20 μl biotinylated and acceptor bead-conjugated antibodies in OptiPlate-384 (all Perkin Elmer). After 2 hours of incubation at RT, 25μl of Streptavidin donor beads were added at RT for 30 minutes followed by detection with the Envision plate reader. Raw values were normalized to transduced (no fibril) control samples per plate.
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7

Inhibitory Effects of Peptides on Stx2a Binding

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The inhibitory effects of a series of shorter MMβA-mono-derived peptides on binding between the Stx2a A-subunit and MMβA-mono were measured using the AlphaScreen assay, as described previously18 (link). Briefly, biotinylated MMβA-mono (30 nM) was incubated with Stx2a A-subunit (20 nM) in the presence of indicated concentrations of a single shorter peptide and specific anti-Stx2a A-subunit monoclonal antibody (originally obtained) in individual wells of an OptiPlate-384 (PerkinElmer, Waltham, MA, USA) for 30 min at room temperature. Samples were then incubated with anti-IgG (protein A) acceptor beads (20 µg/ml; PerkinElmer) for 30 min, followed by incubation with streptavidin donor beads (20 µg/ml; PerkinElmer) for 1 h at room temperature in the dark. The plate was then subjected to excitation at 680 nm, and emission from wells was monitored at 615 nm with the EnVision system (Perkin Elmer). Data were obtained as arbitrary units (AUs) of signal intensity (counts per second). IC50 values were determined by using Image J software ver. 1.53 k.
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8

CFTR-NHERF1 Binding Assay Protocol

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We used the AlphaScreen
(amplified luminescent proximity homogeneous
assay) GST Detection Kit (PerkinElmer; Waltham, MA) to study interactions
of purified full-length Flag-WT CFTR and Flag-ΔF508 CFTR with
the GST-NHERF1-PDZ2 domain. All proteins used were of high quality
and purified under nondenaturing conditions (we tested protein interactions
with syntaxin 1A at the N-terminus and protein binding to Azido ATP;
data not shown). In brief, starting with a 100 nM concentration, CFTR
protein (WT and ΔF508) was serially diluted (in 1/2 log dilution
series) in the assay buffer (1x HEPES, 0.1% BSA, 0.05% Tween 20 [v/v],
pH 7.2) containing GST-NHERF1-PDZ-2 (100 nM final concentration) and
biotin-CFTR-C-tail peptide (100 nM final concentration). The resulting
solutions were incubated at room temperature for 30 min. In triplicate,
each sample solution (15 μL) was transferred to a white opaque
384-well microplate (OptiPlate-384; PerkinElmer) into which anti-GST
acceptor beads (5 μL; 20 μg/mL final concentration) were
added and incubated at room temperature for 30 min. Streptavidin donor
beads (5 μL; 20 μg/mL final concentration) were then added
and incubated at room temperature for 2 h. The plate was read on a
FLUOstar-Omega plate reader (Ortenberg, Germany).
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9

Quantifying SMOX Levels in A549 Cells

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The AlphaLISA assay was setup by identifying suitable antibodies and optimizing antibody concentrations according to the manufacturer’s instructions (see results for details). For the measurement of [SMOX] in A549 cells, cell pellets containing 6 million cells as determined from automated trypan blue cell counting using a Luna (Logos Biosystems) cell counter were resuspended and lysed in 500 μl AlphaLISA buffer followed by centrifugation at 14,000 g. The supernatant was aliquoted and stored at −80 ˚C until use. In the final assay, a SMAB2/ His6-tagged Fab33 mixture was prepared by diluting SMAB2 to 0.5 nM and and His6-Fab33 to 5 nM in 1x AlphaLISA buffer. A volume of 5 μL of this mixture was added to 10 μL of 10x diluted lysate sample (for determinations of SMOX concentration) or 10 μl AlphaLISA buffer containing a known amount of SMOX (for calibration) in a 384 well white assay plate (OptiPlate-384, Perkin Elmer) and incubated for one hour at RT. Then 5 μL 50 μg/ml acceptor beads (AL178C, Perkin Elmer) was added followed by incubation for one hour at RT. For the last step of the assay, 5 μL donor beads (200 μg/mL) (AS105D, Perkin Elmer) was added per well. After one hour incubation at RT in the dark, the Alpha signal was read using an Alpha compatible reader (Synergy NEO, BioTek). Lysate and calibration samples were measured in duplicate.
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10

Screening 5-HT6R Inhibitors via cAMP Assay

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Compounds were examined on
5-HT6R using their ability
to inhibit cAMP production induced by 1 μM (EC80)
5-carboxamidotryptamine (5-CT). The level of cAMP was measured in
1321N1 cells expressing the h5-HT6R (PerkinElmer,
#ES-316-CF). According to the manufacturer’s instructions,
total cAMP was measured using the LANCE cAMP detection kit (PerkinElmer,
#TRF0263). Cells were incubated with a mixture of compounds for 30
min at room temperature (RT) in a white polystyrene OptiPlate-384
(PerkinElmer, #6007299) microplate. After incubation, the reaction
cells were lysed by the addition of 10 μL of cAMP detection
buffer, including Eu-cAMP tracer and ULight-anti-cAMP working solution.
The plate was incubated at RT for 1 h before measuring the signal
with a Tecan multimode plate reader (Infinite M1000 Pro). Compounds
were tested in triplicate at eight concentrations in the range from
10–11 to 10–4 M. Kb constants were calculated from Cheng–Prusoff
equation63 (link) adapted to functional assays.
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