GPCR screening was performed according to the protocol reported by Chen et al.[36 (link)] Briefly, HTLA cells (a HEK293-derived cell line containing a stably integrated tTa-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin were seeded in 96-well tissue culture plates (Eppendorf) and transfected with 200 ng of S1PR4 plasmid (Addgene plasmid #66499)[25 (link)] for 20 h. The medium was then replaced with Dulbecco’s Modified Eagle Medium (DMEM) with 20 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer and 1% Penicillin/Streptomycin, and independently treated with compounds 1 and 2 , sphingosine-1-phosphate (S1P) (Fisher Scientific), and dimethyl sulfoxide (DMSO) solvent vehicle after 2 h. After 20 h of treatment, the luminescence was read by incubating each well for 20 min with 50 μl/well of Bright-Glo solution (Promega) diluted 20-fold in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM-HEPES. The luminescence was measured using Perkin Elmer EnVision 2100 plate reader.
Bright glo solution
Manufactured by Promega
Bright-Glo solution is a luciferase assay reagent used for the quantitative measurement of ATP levels in cells. It is a sensitive and stable chemiluminescent substrate that emits light in the presence of luciferase enzyme and ATP. The intensity of the emitted light is proportional to the ATP concentration, allowing for the determination of cellular ATP levels.
Automatically generated - may contain errors
Lab products found in correlation
96 well tissue culture plates, by Eppendorf (2 mentions)
S1pr4 plasmid, by Addgene (2 mentions)
Sphingosine 1 phosphate (s1p), by Thermo Fisher Scientific (2 mentions)
Envision 2100 plate reader, by PerkinElmer (2 mentions)
Clear bottom 96 well plate, by Thermo Fisher Scientific (2 mentions)
384 well white clear bottom cell culture plates, by Greiner (2 mentions)
Luminoskan ascent 392 microplate luminometer, by Thermo Fisher Scientific (2 mentions)
Spectramax i3x plate reader, by Molecular Devices (1 mentions)
Centro lb 963 microplate luminometer, by Berthold Technologies (1 mentions)
96 well plate, by Greiner (1 mentions)
Spectramax i3 plate reader, by Molecular Devices (1 mentions)
Spectramax plate reader, by Molecular Devices (1 mentions)
Cxcl1, by Thermo Fisher Scientific (1 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions)
Cxcl7, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine max, by Polysciences (1 mentions)
12 protocols using bright glo solution
1
GPCR Screening of S1PR4 Receptor
2
HTLA Cell Transfection and DRD2 Signaling
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HTLA cells
were a gift from the laboratory of G. Barnea and were maintained in
DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL
streptomycin, 2 μg/mL puromycin, 100 μg/mL hygromycin
B, and 100 μg/mL G418 in a humidified atmosphere at 37 °C
in 5% CO2. On day 1, cells were plated at a density of
1 × 105 cells/cm2 in a black wall, clear-bottom
96-well plate (Nunc). The following day (day 2), cells were transfected
with a 10× solution of 3:1 mixture of DRD2-TANGO/Optifect Transfection
Reagent (Thermo) in unsupplemented DMEM. On day 3, 1× drug stimulation
solutions were prepared in filter-sterilized unsupplemented DMEM.
The transfection media was shaken or aspirated from the wells, and
drug stimulation solutions were gently added. On day 4, drug solutions
were removed from one well every 10 s (to maintain consistency of
incubation time) and 50 μL per well of Bright-Glo solution (Promega)
diluted 20-fold in HBSS was added. After incubation for 2 min at room
temperature, luminescence was counted with an integration time of
10 s in a Spectramax i3x plate reader (Molecular Devices).
were a gift from the laboratory of G. Barnea and were maintained in
DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL
streptomycin, 2 μg/mL puromycin, 100 μg/mL hygromycin
B, and 100 μg/mL G418 in a humidified atmosphere at 37 °C
in 5% CO2. On day 1, cells were plated at a density of
1 × 105 cells/cm2 in a black wall, clear-bottom
96-well plate (Nunc). The following day (day 2), cells were transfected
with a 10× solution of 3:1 mixture of DRD2-TANGO/Optifect Transfection
Reagent (Thermo) in unsupplemented DMEM. On day 3, 1× drug stimulation
solutions were prepared in filter-sterilized unsupplemented DMEM.
The transfection media was shaken or aspirated from the wells, and
drug stimulation solutions were gently added. On day 4, drug solutions
were removed from one well every 10 s (to maintain consistency of
incubation time) and 50 μL per well of Bright-Glo solution (Promega)
diluted 20-fold in HBSS was added. After incubation for 2 min at room
temperature, luminescence was counted with an integration time of
10 s in a Spectramax i3x plate reader (Molecular Devices).
3
HTLA Cell-Based Luciferase Assay
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HTLA cells, (an HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) were a gift from the lab of Richard Axel, and were maintained in DMEM supplemented with 10% FBS, 2 μg/ml puromycin and 100 μg/ml hygromycin B in a humidified atmosphere at 37°C in 5% CO2. For transfection, cells were plated at 9 to 10 × 106 cells per 150 mm cell culture dish (day 1). The following day (day 2), cells were transfected using the calcium phosphate method. On day 3, transfected cells were transferred at 15,000 to 20,000 cells per well in 40 μl of medium into poly-L-lysine coated and rinsed 384-well white clear-bottom cell culture plates (Greiner Bio-one). On day 4, 3.5x drug stimulation solutions were prepared in filter-sterilized assay buffer, which consisted of 20 mM HEPES and 1x HBSS at pH 7.4, and 20 μl added to each well. On day 5, medium and drug solutions were removed from the wells (by aspiration or shaking), and 20 μl per well of Bright-Glo solution (Promega) diluted 20-fold in assay buffer were added to each well. After incubation for 15 to 20 minutes at room temperature, luminescence was counted in a Trilux luminescence counter. Results in the form of RLU (relative luminescence units) were exported into Excel spreadsheets, and Graphpad Prism was used for analysis of data. To measure constitutive activity, no ligand was added on day 4.
4
HTLA Cell-Based Luciferase Assay
5
PRESTO-Tango Assay for GPRC5C
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PRESTO-Tango assay was performed according to the protocol in Kroeze et al.36 (link), with slight modifications. Briefly, HTLA cells were cultured in 10% FBS DMEM with 2 μg ml−1 puromycin and 100 μg ml−1 hygromycin B. Cells were transfected with 30 μg ml−1 of the GPRC5C-Tango plasmids using JetPrime. After 24 h of transfection, cells were plated at 60,000–80,000 cells per well into a 96-well plate. Then, 3.5× concentration of the drug was prepared in 20 mM HEPES and 1× HBSS, pH 7.4, and 40 μl of the drug population was added to each well. After 24 h of incubation, cells were washed with PBS and transferred into a 96-well plate (Greiner) with 40 μl of Bright-Glo solution (Promega). Following a 5-min incubation at RT, each well was measured for 5 s on a Centro LB 963 Microplate Luminometer (Berthold).
6
MRGPRX2-mediated Cellular Activation Assay
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HTLA cells stably expressing MRGPRX2 (5×104 cells/well) were plated into a 96-well plate with an antibiotic-free medium and incubated for 6 h to allow cells’ adherence at 37°C with 5% CO2. Following 6 h of incubation, the medium was aspirated, and cells were incubated with C9 (1 or 10 μM) in an antibiotic-free medium for 5 min, followed by stimulation with MRGPRX2 agonist for an additional 16 h at 37°C with a 5% CO2 incubator. After 16 h, the medium was aspirated and replaced with 100 μl of Bright-Glo solution (Promega). The relative luminescence unit was measured in a Thermo Labsystems Luminoskan Ascent 392 Microplate Luminometer (42 (link)). For HTLA cells transiently transfected with MRGPRX2 or its missense variant, V282M tango plasmid, cells treated with C9 (10 μM), C7 (10 μM), or non-treated control and assay were performed similarly (32 (link)).
7
GPCR Screening of S1PR4 Receptor
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GPCR screening was performed according to the protocol reported by Chen et al.[36 (link)] Briefly, HTLA cells (a HEK293-derived cell line containing a stably integrated tTa-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin were seeded in 96-well tissue culture plates (Eppendorf) and transfected with 200 ng of S1PR4 plasmid (Addgene plasmid #66499)[25 (link)] for 20 h. The medium was then replaced with Dulbecco’s Modified Eagle Medium (DMEM) with 20 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer and 1% Penicillin/Streptomycin, and independently treated with compounds 1 and 2 , sphingosine-1-phosphate (S1P) (Fisher Scientific), and dimethyl sulfoxide (DMSO) solvent vehicle after 2 h. After 20 h of treatment, the luminescence was read by incubating each well for 20 min with 50 μl/well of Bright-Glo solution (Promega) diluted 20-fold in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM-HEPES. The luminescence was measured using Perkin Elmer EnVision 2100 plate reader.
8
MRGPRX2 Ligand Activation Assay
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HTLA cells expressing either WT MRGPRX2 or its variants (5 × 104 cells per well) were plated into a 96-well plate in triplicates in a total volume of 160 μL antibiotic-free medium and incubated for 6 h at 37 °C to allow attachment. After 6 h, the medium was aspirated, and cells were incubated with MRGPRX2 ligands in 160 μL antibiotic-free medium for additional 16 h at 37 °C. The medium and ligands were then aspirated and 100 μL of Bright-Glo solution (Promega) was added to each well. Relative luminescence unit was measured in a Thermo Labsystems Luminoskan Ascent 392 Microplate Luminometer [20 (link),25 (link)].
9
PRESTO-Tango GPCR-ome Screening Protocol
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The three peptides from human microbiota were synthesized by GenScript Biotech, New Jersey. Screening of compounds in the PRESTO-Tango GPCR-ome was accomplished using previously described methods with several modifications57 (link). First, HTLA cells were plated in DMEM with 2% dialyzed FBS and 10 U/mL penicillin-streptomycin. Next, the cells were transfected using an in-plate PEI method70 . PRESTO-Tango receptor DNAs were resuspended in OptiMEM and hybridized with PEI prior to dilution and distribution into 384-well plates and subsequent addition to cells. After overnight incubation, drugs diluted in DMEM with 1% dialyzed FBS were added to cells without replacement of the medium. After another overnight incubation, the culture medium and peptide-including buffer were removed by aspiration. 20 μl of Bright-Glo solution (Promega) diluted 20-fold in assay buffer was added into each well. The cells were incubated with buffer at room temperature for 15-20 minutes. Then, the luminescence was measured in a Trilux luminescence counter. The relative luminescence units (RLU) were collected from the machine and calculated by fold change based on the basal RLU.
10
HTLA Cell Transfection and Luminescence Assay
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HTLA cells
were a gift from the laboratory of G. Barnea and were maintained in
DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin
and 100 μg/mL streptomycin, 2 μg/mL puromycin, 100 μg/mL
hygromycin B, and 100 μg/mL G418, in a humidified atmosphere
at 37 °C in 5% CO2. On day 1, cells were plated at
a density of 1x105 cells/cm2 in a black wall,
clear bottom 96 well plate (Nunc). On the following day (day 2), cells
were transfected with a 10× solution of 3:1 mixture of HTR2C-TANGO
(Addgene #66411):Optifect Transfection Reagent (Thermo) in un-supplemented
DMEM. On day 3, 1× drug stimulation solutions were prepared in
filter-sterilized unupplemented DMEM. The transfection media was shaken
or aspirated from the wells, and drug stimulation solutions were gently
added. On day 4, drug solutions were removed from one well every 10
s (to maintain consistency of incubation time) and 50 μL per
well of Bright-Glo solution (Promega) diluted 20-fold in HBSS was
added. After incubation for 2 min at room temperature, luminescence
was counted with an integration time of 10 s in a Spectramax i3×
plate reader (Molecular Devices). Drug concentrations were experimentally
measured in triplicate. Statistical analysis was performed using GraphPad
Prism 9.
were a gift from the laboratory of G. Barnea and were maintained in
DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin
and 100 μg/mL streptomycin, 2 μg/mL puromycin, 100 μg/mL
hygromycin B, and 100 μg/mL G418, in a humidified atmosphere
at 37 °C in 5% CO2. On day 1, cells were plated at
a density of 1x105 cells/cm2 in a black wall,
clear bottom 96 well plate (Nunc). On the following day (day 2), cells
were transfected with a 10× solution of 3:1 mixture of HTR2C-TANGO
(Addgene #66411):Optifect Transfection Reagent (Thermo) in un-supplemented
DMEM. On day 3, 1× drug stimulation solutions were prepared in
filter-sterilized unupplemented DMEM. The transfection media was shaken
or aspirated from the wells, and drug stimulation solutions were gently
added. On day 4, drug solutions were removed from one well every 10
s (to maintain consistency of incubation time) and 50 μL per
well of Bright-Glo solution (Promega) diluted 20-fold in HBSS was
added. After incubation for 2 min at room temperature, luminescence
was counted with an integration time of 10 s in a Spectramax i3×
plate reader (Molecular Devices). Drug concentrations were experimentally
measured in triplicate. Statistical analysis was performed using GraphPad
Prism 9.
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