To study HIF signaling, a HCT116 reporter cell line was used that is transfected with a 598-pGL3-HIF-RE-Luc reporter plasmid that contains a luciferase gene expressed under the control of a VEGF promoter-derived HRE.41 (link) Cells were selected using 100 μg/ml Hygromycin B (Invitrogen). Luciferase activity was measured using the luminescence kit Steady-Glo (Promega, Fitchburg, WI, USA) and a luminescence plate reader (PHERAstar (BMG Labtech, Ortenberg, Germany)) according to the manufacturer's instructions. Briefly, HCT 116-HRE-Luc cells were plated at 3000 cells/5 μl in white 384-well small volume plates (Greiner) using a liquid dispenser (Multidrop Combi, Thermo Scientific), 5 μl compounds were added and cells were either incubated in a normal incubator or in a hypoxia chamber at >1% O2 for 16 to 24 h. In all, 5 μl SteadyGlo (Promega) were then added to cells and incubated for 30 min in the dark. Subsequently, luminescence intensity was measured in a plate reader.
Steady glo
Manufactured by Promega
Sourced in United States
Steady-Glo is a luciferase-based reagent system designed to provide a stable, sustained signal for luminescent detection applications. It is suitable for use with a variety of sample types and detection platforms.
Automatically generated - may contain errors
Lab products found in correlation
M1000 plate reader, by Tecan (7 mentions)
Polybrene, by Merck Group (5 mentions)
Model n4, by Agilent Technologies (4 mentions)
Cell lysis buffer, by Promega (4 mentions)
Modulus 2 microplate reader, by Promega (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Topcount, by PerkinElmer (3 mentions)
Prism 7, by GraphPad (3 mentions)
Nivo spectrophotometer, by PerkinElmer (3 mentions)
Glomax microplate reader, by Promega (2 mentions)
White flat bottomed 96 well plate, by Jet Biofil (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Recombinant mil 1β, by R&D Systems (2 mentions)
Mil 36γ, by R&D Systems (2 mentions)
Recombinant hil 1β, by R&D Systems (2 mentions)
Mil 33, by R&D Systems (2 mentions)
Celltiter glo, by Promega (2 mentions)
Recombinant human il 1α or il 1β, by R&D Systems (2 mentions)
White flat bottom polystyrene tissue culture treated microplates, by Corning (2 mentions)
76 protocols using steady glo
1
HIF Signaling Reporter Assay
2
Dual Luciferase Assay for IRES Activity
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After BHK-21 cells were knocked down or overexpressed, the cells were transfected with the corresponding bicistronic reporter plasmid, psiCHECK or psiCHECK-FMDV, respectively. At 24 h post-transfection, the cells were lysed by the special cell lysate buffer for dual luciferase reporter assay, and the signal intensities of Firefly luciferase (Fluc) and Renilla luciferase (RLuc) were detected with the Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions. Monocistronic luciferase activity was measured in the rabbit reticulocyte in vitro translation system [rabbit reticulocyte lysate (RRL), Promega] as previously described (29 (link)). The template with pGL3-FMDV IRES-FLuc gene for in vitro translation was synthesized by Azenta Life Sciences (Suzhou, China). RALY (50 pmol) or a non-specific control protein, BSA, and 0.5 µg of template RNA in vitro transcript were added to 200 µL of the reaction system and incubated at 30°C for 40 min, and luciferase activity was immediately determined with Steady-Glo (Promega).
3
Virus Infection Quantification in TZM-bl Cells
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Virus-containing supernatants were precleared via low-speed centrifugation (5 min, 1500 rpm) and filtered through a 0.45 µm syringe filter. The amounts of virus in the supernatants were quantified using an RT assay. TZM-bl indicator cells (CD4+, CCR5+, CXCR4+) were plated in a 24-well plate (1 mL; 5 × 104 cells/well) and infected with 50 to 150 µL of viral supernatant. Typically, infections were performed in triplicate. After 48 h, the culture medium was aspirated, and cells were lysed in the wells with 200 µL of lysis buffer (25 mM Tris-HCl pH 7.8, 8.0 mM MgCl2, 1.0 mM DTT, 1.0% TritonX-100, 15% glycerol). Luciferase activity was determined by combining 10 µL of each lysate with 30 µL of luciferase substrate (Steady-Glo; Promega Corp., Madison, WI, USA). The light emission was measured using a GloMax microplate reader (Promega). The results were corrected for differences in the input virus.
4
DnaK-Assisted Luciferase Refolding Assay
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DnaK-assisted luciferase refolding assays were performed as described previously (39 (link)) with slight modifications. The refolding of guanidine hydrochloride-denatured firefly luciferase (Promega) was performed in Buffer B containing 1 mm ATP without DTT for 1 h at 37 °C. DnaK, DnaJ, and GrpE were added into the refolding system at final concentrations of 800, 160, and 400 nm , respectively. Each reaction was performed in triplicate, and 5 μl of the refolding mixture was removed and added to a white flat bottomed 96-well plate (JET Biofil) that was preloaded with 10 μl of Steady-Glo (Promega). After mixing, the luminescence was measured on a SpectraMax M3e multimode plate reader using a 500-ms integration time.
5
Estrogen Receptor Antagonist Screening
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7dF3
cells were seeded
at 5000 per well in 50 μL of Dulbecco’s modified Eagle’s
medium (DMEM) with 4 mM supplementall -glutamine in 384-well
white tissue culture plates (Corning 3570). After incubating overnight
at 37 °C/5% CO2, compounds ranging in final concentration
from 90 μM to 0.3 nM were added in duplicate to the wells. After
2 h of further incubation as above, β-oestradiol was added to
a final concentration of 10 μM. The cells were incubated for
24 h at 37 °C/5% CO2 and then 25 μL of luciferase
reagent (SteadyGlo, Promega) was added, and the cells were mixed.
After leaving the plate for 60 min at room temperature, luminescence
was read on a plate luminescence reader (TopCount, PerkinElmer). The
percentage inhibition for each compound concentration was calculated
using total counts (no compound) and low counts (no β-oestradiol)
as highs and lows, respectively. IC50 was subsequently
determined using a curve-fitting software package in Excel (Excelfit,
IDBS).
cells were seeded
at 5000 per well in 50 μL of Dulbecco’s modified Eagle’s
medium (DMEM) with 4 mM supplemental
white tissue culture plates (Corning 3570). After incubating overnight
at 37 °C/5% CO2, compounds ranging in final concentration
from 90 μM to 0.3 nM were added in duplicate to the wells. After
2 h of further incubation as above, β-oestradiol was added to
a final concentration of 10 μM. The cells were incubated for
24 h at 37 °C/5% CO2 and then 25 μL of luciferase
reagent (SteadyGlo, Promega) was added, and the cells were mixed.
After leaving the plate for 60 min at room temperature, luminescence
was read on a plate luminescence reader (TopCount, PerkinElmer). The
percentage inhibition for each compound concentration was calculated
using total counts (no compound) and low counts (no β-oestradiol)
as highs and lows, respectively. IC50 was subsequently
determined using a curve-fitting software package in Excel (Excelfit,
IDBS).
6
Quantifying HCV Replication Levels
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We used four HCV subgenomic replicon cell lines, FLR3-1 (genotype 1b, Con-1)47 (link), R6FLR-N (genotype 1b, strain N)26 (link), JFH-1/FLR/K4 (genotype 2a)48 (link), and RMT-tri (genotype 1a)49 (link), which have the firefly luciferase gene for the sensitive and precise quantification of the HCV replication levels using a luciferase assay. We also used REF cells30 (link) which harbor the divided-full genome replicon for analysis of the HCV 5′ UTR sequences. Each cell line was seeded at a density of 5 × 103 per well in 96-well tissue culture plates, and grown (at 37°C and 5% CO2) in complete Dulbecco's modified Eagle's medium supplemented with Glutamax I (Invitrogen, Carlsbad, CA) and containing 5% fetal calf serum (Invitrogen). Cells were transfected with 30 nM siRNA using RNAiMax (Invitrogen, Carlsbad, CA). After 72 hours, luciferase activity was determined in triplicate using the Steady-Glo or Bright-Glo luciferase assay kit (Promega Madison, WI). The luciferase signal was measured using an LB940 luminometer (Berthold, Freiburg, Germany) and the results were expressed as the mean percentage of control. IC50 values of siRNA were calculated by nonlinear curve-fitting using the equation: Y = 100-(YBottom × X/(IC50 + X)), where Y represents percent inhibition and X represents the concentration of siRNAs.
7
Inhibiting HIV-1 CXCR4 Binding in TZM-bl Cells
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An inhibitor that blocks HIV-1 binding to CXCR4 (1 μM AMD3100, Sigma-Aldrich) was added to TZM-bl cells for 1 h at 37°C before infection with HIV-1 NL4.3. GML was added at the time of HIV-1 inoculation, and cells were maintained in the inhibitor for the duration of the infection. Infection of TZM-bl cells was assessed after 24 h by RLU luciferase assay (Steady-Glo; Promega).
8
Assessing PSNP Effects on Gluconeogenesis
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To assess the effect of PSNPs on gluconeogenesis, Tg(pck1:Luc2) larvae were exposed to 0, 0.2, 2, and 20 mg L−1 PSNPs in 48-well plates with 1 mL exposure volume per well. Co-exposure to 40 mM glucose and 1 µM mifepristone was performed as well (1 larva per well). In the Tg(pck1:Luc2)57 (link) fish, the cytosolic phosphoenolpyruvate carboxykinase (pck1) promoter is labelled with firefly luciferase gene luc2. Phosphoenolpyruvate is used as the starting substrate for gluconeogenesis and transcriptional alterations of pck1 are predominantly expressed in the liver and kidneys. At 120 hpf, larvae were washed twice in egg water and three larvae were pooled to one replicate with five replicates sampled per group. Larvae were lysed in a final volume of 100 µL of egg water using a point sonicator (Qsonica) with five pulses at an amplitude of 20% and stored on ice until centrifugation at 13,000 r.p.m. for 3 min at 4 °C. Subsequently, 90 µL of the supernatant was transferred to a 96-well plate (Thermo Fisher, flat bottom, white) and 50 µL of Steady-Glo (Promega) was added. The plate was incubated in the dark for 1 h after which the bioluminescence was quantified using a plate reader (Tecan Infinite M1000 PRO).
9
SARS-CoV-2 Spike Protein Neutralization Assay
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Full-length SARS-CoV-2 S protein was used to create pseudo-typed lentivirus as described elsewhere [15 ], with slight modifications. Plasmids encoding S and human immunodeficiency virus type 1 structural genes were cotransfected into 293T cells using PEI Max and grown at 32°C for 3 days before harvesting supernatants. Next, 293T cells stably expressing ACE2 were plated in 96-well plates at a density of 104 per well 24 hours before viral challenge, and polybrene was added at 2 ug/mL for 30 minutes before viral challenge.
Heat-inactivated plasma samples from COVID-19–positive patients or pre–COVID-19 controls were serially diluted from 1:50–1:109 350 and coincubated with virus for 60 minutes before plating on cells. The plates were incubated at 37°C for 65 hours, the media removed, and 100 μl of Steady Glo (Promega) added. Relative light unit (RLU) values were read on a luminometer, and the percentage of neutralization was calculated as (RLUvirus − RLUvirus+plasma)/(RLUvirus) × 100. We selected a subset of total samples to span the range of inhibition for both COVID-19–positive and control samples, and all samples analyzed are reported. The 50% inhibitory concentration (IC50) values were extrapolated from a nonlinear regression model and represent the reciprocal plasma dilution at which 50% viral inhibition was recorded.
Heat-inactivated plasma samples from COVID-19–positive patients or pre–COVID-19 controls were serially diluted from 1:50–1:109 350 and coincubated with virus for 60 minutes before plating on cells. The plates were incubated at 37°C for 65 hours, the media removed, and 100 μl of Steady Glo (Promega) added. Relative light unit (RLU) values were read on a luminometer, and the percentage of neutralization was calculated as (RLUvirus − RLUvirus+plasma)/(RLUvirus) × 100. We selected a subset of total samples to span the range of inhibition for both COVID-19–positive and control samples, and all samples analyzed are reported. The 50% inhibitory concentration (IC50) values were extrapolated from a nonlinear regression model and represent the reciprocal plasma dilution at which 50% viral inhibition was recorded.
10
Reporter-based Screening of GFL Receptor Activators
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To identify compounds activating GFL receptors and to check their ability to activate intracellular signaling via TrkA and TrkB, we used previously developed reporter-gene based systems (Sidorova et al., 2010 (link)). MG87 murine fibroblasts were stably transfected with Pathdetect Elk-1 and one of the following: TrkA, TrkB, RET, GFRα1/RET, GFRα3/RET. The day before the experiment reporter cells were plated into 96- or 384-well cell culture plates (PerkinElmer) at 175,000–200,000 cell/ml density in DMEM, 10% FBS, 100 μg/ml Normocin (Invivogen, Cat# ant-nr-1), 1% DMSO, 15 mM Hepes pH 7.2. The next day compounds or proteins under study were applied to the cells in desirable concentration. The following day the cells were lysed and luciferase activity was measured using a luciferase detection reagent (SteadyGlo, Cat# E2550, or Luciferase assay reagent, Cat# E1501, Promega). The luminescence was measured using a plate luminometer (Micorbeta-2 or TopCount, PerkinElmer). During high throughput screening each compound was tested in a single concentration (5 μM) and a single repeat. Hit confirmation experiments were performed in triplicates. Dose-dependent studies in all reporter cell lines were made in quadruplicates. EC50 was calculated based on the results of at least three independent experiments.
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