293A cells harboring a HiBiT tag sequence25 (link) in the N-terminus of the BRD4 gene (293A_HiBiT-BRD4) were established using the CRISPR-Cas9 system in our laboratory. Four thousand cells per well were plated on a 384-well white plate (Greiner). The next day, cells were treated with DMSO or BET protein degraders at the indicated concentrations and incubated for 3, 8 and 24 h. After incubation, cells were treated with Nano Glo HiBiT Lytic Detection System (Promega) to detect the HiBiT-BRD4-derived luminescent signals with EnVision Xcite (PerkinElmer). The number of cells in each well was estimated based on DNA content measured using CellTox Green Reagent (Promega) with EnVision Xcite and the luminescent intensity of HiBiT-BRD4 was normalized with the fluorescent signals of CellTox Green Reagent. The BRD4 expression levels in BET protein degrader-treated cells were calculated as the percentage of those in DMSO-treated cells.
White 384 well plate
Manufactured by Greiner
Sourced in Germany, Austria
The white 384-well plates are a type of laboratory equipment used for various experimental and analytical purposes. These plates have 384 individual wells, each designed to hold a small volume of sample or reagent. The white color of the plates helps to enhance visibility and contrast during visual inspections or when using optical detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Envision plate reader, by PerkinElmer (6 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Pathhunter β arrestin 2 assay, by Eurofins (2 mentions)
Pherastar fsx plate reader, by BMG Labtech (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Plate washer, by Thermo Fisher Scientific (2 mentions)
Prism 5, by GraphPad (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Emem 10 009 cv, by Corning (2 mentions)
Complete ultra protease inhibitor, by Roche (2 mentions)
Nano glo hibit lytic detection system, by Promega (1 mentions)
Envision xcite, by PerkinElmer (1 mentions)
Celltox green reagent, by Promega (1 mentions)
Prism software, by GraphPad (1 mentions)
Safire2 plate reader, by Tecan (1 mentions)
Atplite 1 step kit, by PerkinElmer (1 mentions)
Camptothecin cpt, by Cayman Chemical (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
Prism 7, by GraphPad (1 mentions)
26 protocols using white 384 well plate
1
Quantifying BRD4 Degradation by BET Inhibitors
2
Luminescent and Colorimetric Assays for Thioredoxin Reductase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Compounds from 10 mM stock solutions were transferred to assay plates by acoustic transfer (EDC Biosystems, Milmont, CA). The TbTR luminescent assay was performed in 384-well white plate (Greiner Bio One, Frickenhausen, Germany). The following components were added to the plates to a final volume of 30 μL: 0.1 nM TR, 20 μM NADPH, 10 μM TS2 in 50 mM HEPES (pH 7.4), 40 mM NaCl, 0.01% BSA. After 60 min of incubation at room temperature the residual amount of NADPH was measured by addition of an equal volume of NADPH-Glo as per the manufacturer’s protocol and the luminescent signal was acquired by an EnVision plate reader (PerkinElmer, Waltham, MA). The DTNB assay was performed in a final volume of 50 μl by addition of 2 nM TR, 100 μM NADPH, 4 μM TS2 and 200 μM DTNB in 40 mM HEPES (pH 7.4), 1 mM EDTA, 0.01% BSA and 0.05% Tween-20. After 10 minutes of incubation at room temperature the absorbance signal (412 nm) was detected using the Safire2 plate reader (Tecan, Switzerland). The human glutathione reductase (hGR) activity assay was carried out as described by Turcano et al.[23 (link)]. Results were analyzed using Prism software (GraphPad, San Diego, CA) and Vortex (Dotmatics, Bioshops Stortford, UK). Dose-response curves were fitted by four-parameter logistic regression.
3
Quantifying β-Arrestin-2 Recruitment in MOR-Expressing Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The
measurement of hMOR-stimulated β-arrestin-2 recruitment was
performed by the PathHunter β-arrestin-2 assay (DiscoverX, Birmingham,
UK) according to the published procedure.25 (link) U2OS cells stably coexpressing the human MOR and the enzyme acceptor
(EA)-tagged β-arrestin-2 fusion protein (U2OS-hMOR-β-arrestin-2
cells) were seeded in the cell plating medium into 384-well white
plates (Greiner Bio-One, Kremsmünster, Austria) at a density
of 5000 cells in 20 μL per well and maintained at 37 °C
for 24 h. After incubation with various concentrations of test peptides
in PBS for 90 min at 37 °C, the detection mix was added, and
incubation was continued for additional 60 min at room temperature.
Chemiluminescence was measured with the PHERAstar FSX plate reader
(BMG LABTECH, Germany). All experiments were performed in duplicate
and repeated three times with independently prepared samples.
measurement of hMOR-stimulated β-arrestin-2 recruitment was
performed by the PathHunter β-arrestin-2 assay (DiscoverX, Birmingham,
UK) according to the published procedure.25 (link) U2OS cells stably coexpressing the human MOR and the enzyme acceptor
(EA)-tagged β-arrestin-2 fusion protein (U2OS-hMOR-β-arrestin-2
cells) were seeded in the cell plating medium into 384-well white
plates (Greiner Bio-One, Kremsmünster, Austria) at a density
of 5000 cells in 20 μL per well and maintained at 37 °C
for 24 h. After incubation with various concentrations of test peptides
in PBS for 90 min at 37 °C, the detection mix was added, and
incubation was continued for additional 60 min at room temperature.
Chemiluminescence was measured with the PHERAstar FSX plate reader
(BMG LABTECH, Germany). All experiments were performed in duplicate
and repeated three times with independently prepared samples.
4
Cytotoxicity Evaluation of Camptothecin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated in 384-well white plates (Greiner) at 4,000 cells/well in 40 μL complete medium with various concentrations of camptothecin (CPT; Cayman Chemical). Viability was determined using ATPlite 1-step kits (PerkinElmer). After 72 h, ATPlite solution was added (20 μL/well), and luminescence was then measured with an Infinite M200 Microplate Reader (TECAN). The ATP level in untreated cells was defined as 100%. The viability (%) of treated cells was defined as ATP-treated cells/untreated cells × 100. The data were transferred to the GraphPad Prism 7 software, and graphs were created.
5
Cell Viability Assay for RUVBL1/2 Inhibitors
6
Quantifying hMOR-Mediated β-Arrestin2 Recruitment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The measurement of hMOR stimulated β-arrestin2 recruitment was performed using the PathHunter® β-arrestin2 assay (DiscoveRx, Birmingham, UK) according to the published procedure [20 (link)]. U2OS cells that stably co-expressed the human MOR and the enzyme acceptor (EA) tagged β-arrestin2 fusion protein (U2OS-hMOR-βarrestin2 cells) were seeded in cell plating medium into 384-well white plates (Greiner Bio-One, Kremsmünster, Austria) at a density of 5000 cells in 20 μL per well, and maintained at 37 °C for 24 h. After incubation with various concentrations of the test compounds in phosphate buffered saline (PBS) for 90 min at 37 °C, the detection mix was added, and incubation was continued for an additional 60 min at room temperature. Chemiluminescence was measured with the PHERAstar FSX plate reader (BMG Labtech, Germany). All experiments were performed in duplicate and repeated three times with independently prepared samples.
7
NMT Inhibitor Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in 384-well white plates (Greiner) at 375 to 1,500 cells per well according to the linear relationship measured from a standard curve of each cell line. 24 h after seeding, cells were treated with NMT inhibitor (three fold dilution, eight dilution points, in duplicate). After 72 h of treatment, cell viability was measured using CellTiter Glo Luminescent Cell Viability Assay (Promega), according to the manufacturer’s description, and IC50 values were calculated using the percentage of growth of treated cells versus the DMSO control with GraphPad Prism 7.0 software.
8
Quantitative Compound Screening in MEFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
50 nl of test compounds (2 mM solution in DMSO) were transferred to tissue culture-treated, 384-well white plates (Greiner Bio One, Frickenhausen, Germany) by acoustic transfer (EDC biosystems, Milmont, CA, USA). 20 μl of a suspension of 104Tbx1lacZ/+ MEF cells were added to assay plates. After 48 h incubation at 37 °C, 5% CO2 in humidified atmosphere and 30 min incubation at RT, we added 20 μl/well of Beta Glo (Beta Glo Assay System, Promega). After 1 h incubation at RT, the signal intensity was quantified by the ViewLux uHTS microplate imager (PerkinElmer, USA). Data analysis was performed using the Dotmatics suite (Dotmatics, Bishops Stortford, UK).
‘Compound A’ (1-(benzo[b]thiophen-3-yl)-3-((5-methylisoxazol-3-yl)methyl)urea) was purchased from Maybridge (Product Code: HTS12348, ACD Code: MFCD04110438).
The purity of the compound was assessed by UPLC analysis to be 91%. A Waters UPLC system with both diode array detection and electrospray (+’ve and –‘ve ion) MS detection was used. The stationary phase was a Waters Acquity UPLC BEH C18 1.7um 2.1x50mm column. The mobile phase was H2O containing 0.1% formic acid or MeCN containing 0.1% formic acid. Flow rate 0.5 mL/min. Sample concentration: 1 mg/mL. Injection volume 2 μl.
‘Compound A’ (1-(benzo[b]thiophen-3-yl)-3-((5-methylisoxazol-3-yl)methyl)urea) was purchased from Maybridge (Product Code: HTS12348, ACD Code: MFCD04110438).
The purity of the compound was assessed by UPLC analysis to be 91%. A Waters UPLC system with both diode array detection and electrospray (+’ve and –‘ve ion) MS detection was used. The stationary phase was a Waters Acquity UPLC BEH C18 1.7um 2.1x50mm column. The mobile phase was H2O containing 0.1% formic acid or MeCN containing 0.1% formic acid. Flow rate 0.5 mL/min. Sample concentration: 1 mg/mL. Injection volume 2 μl.
9
Cell Viability Assay with Dose-Response
10
Quantifying ΔF508-CFTR Trafficking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CFBE41o– TetON cells expressing HRP tagged
ΔF508-CFTR were seeded in white 384-well plates (Greiner) at
a density of 2000 cells per well. Medium containing 500 ng/mL doxycycline
was used to induce expression of ΔF508-CFTR-HRP. After 3 days,
cells were treated with corrector or potentiator compounds and transferred
to an incubator at 33 °C. On day 4, cells were washed five times
with PBS containing Ca2+ and Mg2+ using a Bio-Tek
plate washer and incubated with a chemiluminescent HRP substrate (SuperSignal
West Pico Chemiluminescent Substrate, Thermo Scientific) for 15 min.
Chemiluminescence was measured using an Envision plate reader (PerkinElmer).
Dose response data was fitted using a 4 parameter hill function of
the form Response = neg control + (pos control – neg control)/(1
+ 10(logEC50 -concentration)/HillSlope) to determine EC50 values. Percentage efficacy
was calculated using the following formula: (max
response – neg control)/(C1 response – neg control).
ΔF508-CFTR were seeded in white 384-well plates (Greiner) at
a density of 2000 cells per well. Medium containing 500 ng/mL doxycycline
was used to induce expression of ΔF508-CFTR-HRP. After 3 days,
cells were treated with corrector or potentiator compounds and transferred
to an incubator at 33 °C. On day 4, cells were washed five times
with PBS containing Ca2+ and Mg2+ using a Bio-Tek
plate washer and incubated with a chemiluminescent HRP substrate (SuperSignal
West Pico Chemiluminescent Substrate, Thermo Scientific) for 15 min.
Chemiluminescence was measured using an Envision plate reader (PerkinElmer).
Dose response data was fitted using a 4 parameter hill function of
the form Response = neg control + (pos control – neg control)/(1
+ 10(log
was calculated using the following formula: (max
response – neg control)/(C1 response – neg control).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!