The LUC assay is based on the measurement of the bioluminescent activity of luciferase-expressing NF54Pfs16 gametocytes. The assay was carried out as previously described36 (link), with modifications. Briefly, mature stage V gametocytes on day 12 of gametocytogenesis were seeded in 384 wells white luminescence plates (Culturplate, PerkinElmer) at 0.1% hct and 10% gametocytemia in 45 μl and incubated with compounds for 24 h. In each assay plate, 7 wells containing the gametocytocidal reference compound methylene blue at 10 μM and 7 wells treated only with the solvent 0.4% DMSO were used as in-plate positive and negative controls, respectively. At the end of the incubation, 25 μl medium were aspirated simultaneously from each well and replaced with 15 μl of the homogeneous luciferase reporter gene assay system Steadylite plus (PerkinElmer) without disturbing the settled red blood cells (RBCs), as per our standard method24 (link)36 (link). Luminescence was measured after 1 hr incubation at room temperature using a MicroBeta Trilux (PerkinElmer) multidetector luminometer.
Steadylite plus
Manufactured by PerkinElmer
Sourced in United States, Germany, Sweden
The Steadylite plus is a luminescence detection reagent for use in luminescence-based assays. It provides a stable luminescent signal for the detection of reporter genes such as firefly luciferase and Renilla luciferase.
Automatically generated - may contain errors
Lab products found in correlation
Microbeta 1450 jet luminometer, by PerkinElmer (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Culturplate, by PerkinElmer (1 mentions)
Microbeta trilux, by PerkinElmer (1 mentions)
Filtermax f5, by Molecular Devices (1 mentions)
Culturplate 96, by PerkinElmer (1 mentions)
Tristar, by Berthold Technologies (1 mentions)
N hexane, by Avantor (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
2 3 7 8 tetrachlorodibenzo p dioxin tcdd standard, by AccuStandard (1 mentions)
Cellstar microplates, by Greiner (1 mentions)
Envision multimode reader, by PerkinElmer (1 mentions)
Gw4064, by Merck Group (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Second strand synthesis module, by New England Biolabs (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
E gel ex agarose gel, by Thermo Fisher Scientific (1 mentions)
Stbl3 cells, by Thermo Fisher Scientific (1 mentions)
Herculase 2 phusion polymerase, by Agilent Technologies (1 mentions)
Peg 8000, by Merck Group (1 mentions)
16 protocols using steadylite plus
1
Luciferase-Based Gametocyte Assay
2
Coculture Impacts on Ovarian Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Luciferase‐expressing SKOV3 and ES2 cells were either cultured alone in 96‐well culture plates (1 × 103 cells/well) or were cocultured with iPS‐ML/IFN‐β (5 × 102 to 5 × 103 cells/well). SKOV3 (1 × 103 cells/well) in 96‐well culture plates were cultured alone or were cocultured with ordinary type iPS‐ML (without production of IFN‐β, 1 × 103 cells/well) in the presence or absence of 30 ng/mL recombinant human IFN‐β. Three days later, luciferase substrate (SteadyLite Plus; Perkin‐Elmer, Waltham, MA, USA) was added to the wells, and luciferase activity was measured using a microplate reader (FilterMAX F5; Molecular Devices).
3
Measuring ADGRD1 Receptor Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Because ADGRD1 is a Gs-coupled receptor function was measured with cAMP response element (CRE) secreted alkaline phosphatase (SeAP) and CRE binding protein (CREB) luciferase assays. SeAP plasmid (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) and wt or mutant ADGRD1 constructs were co-transfected with each 75 ng of DNA/well. Basal and peptide agonist induced luciferase as well as SeAP levels were measured after 5 h of incubation. For SeAP experiments endogenously expressed alkaline phosphatases were heat inactivated for 2 h right before the measurement. 10 μM forskolin was used as positive control for CRE and CREB activities. As an additional readout for cAMP formation we measured luciferase activity under the control of CREB. Thus, co-transfection of 50 ng receptor plasmid and 50 ng luciferase plasmid (PathDetect trans-reporting System) was performed using HEK293T cells. Luciferase activity was detected using steadylite plus (PerkinElmer, Rodgau, Germany).
4
Neutralizing Antibody Titer Assay for Canine Distemper Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2 × 104 293-dogSLAM cells were plated into each well of a 96-well white flat-bottomed plate (Culturplate-96, Perkin Elmer, Coventry, UK). Four-fold serum dilutions were prepared in triplicate in complete medium ranging from 1:8 to 1:32768. The diluted serum samples were then added to the 293-dogSLAM cells followed by 2.5 × 103 TCID50 of VSVΔG(CDV) pseudotype. Plates were incubated for 48–72 h at 37 °C, at which time luciferase substrate was added (Steadylite plus™, Perkin Elmer) and the signal analysed on a Microbeta 1450 Jet luminometer (Perkin Elmer). Antibody titres were calculated by interpolating the point at which there was a 90% reduction in luciferase activity (90% neutralisation, inhibitory concentration 90 or IC90) [52] (link).
5
Colon26 Cells Cytokine Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Colon26 cells expressing firefly luciferase (Colon26/Luc)34 were cultured in the presence of recombinant mouse (rm) IFN‐β (10 ng/mL) and/or rmIFN‐γ (10 ng/mL). After 3 d, luciferase substrate (Steadylite plus, Perkin‐Elmer) was added, and luminescence activity was quantified using a microplate reader (TriStar; Berthold Technologies). To detect apoptosis, the cells were stained with annexin V‐fluorescein isothiocyanate (FITC). To assess live cells based on nicotinamide adenine dinucleotide (NADH) quantification using the water‐soluble disulfonated tetrazolium salt, the Cell Counting Kit‐8 (CCK‐8; Dojindo Molecular Technologies) was used. To analyze proliferative activity, cells were centrifuged at 270 g for 6 min, fixed with cold 70% ethanol, incubated for 1 h at −20°C, and stained with an anti‐Ki67 antibody (clone REA183; Miltenyi Biotec).
6
Quantitative TCDD Determination Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dimethyl sulfoxide (DMSO) (99.9%) was purchased from Sigma Aldrich (Stockholm, Sweden) and n-hexane (≥ 98%) was purchased from VWR (Stockholm, Sweden). Steady Lite plus™ was purchased from Perkin Elmer (Hägersten, Sweden). The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard, with a purity of 99.1%, was from AccuStandard Inc. (New Haven, USA).
For more information regarding chemical standards, refer to the Supporting Information (SI).
For more information regarding chemical standards, refer to the Supporting Information (SI).
7
Lentiviral Transduction and Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfer of the compounds with the Echo550 and cell seeding (1200 per well) were performed as above, this time in white 384-well CellStar microplates (Greiner BioOne, Frieckenhausen, Germany). Again, 25 µL of lentiviral supernatant was added before incubating the plates for 3 days. Lentiviral vector titers were adjusted to obtain ca. 50% of the maximal signal, which resulted in a 10-fold window between signal and background. Higher signal intensities were avoided as they would need higher viral titers which would lead to viral toxicity mediated by multiple vector copies per cell. Luciferase activity was measured using SteadyLite plus (PerkinElmer), which was prepared following the manufacturer’s protocol. Briefly, 25 µL of the reagent were added per well, incubated in the dark for 10 min, and luminescence intensity was measured using an Envision multimode reader (PerkinElmer).
8
Transcriptional Regulation Assay in Huh7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Huh7 cells seeded into 96‐well plates were transfected with 10.4 ng plasmid circular DNA (pcDNA)‐retinoid X receptor, 10.4 ng pcDNA‐FXRα2/pcDNA3.1 together with 10.4 ng and 40 ng pGL3‐IBAP‐Luc and pcDNA3.1‐green fluorescent protein using Fugene 6 transfection reagent (Promega) at a 3:1 ratio. Twenty‐four hours later, cells were washed and treated with 0 or 50 μM compound ± 0.5 μM GW4064 (Sigma‐Aldrich). After 24 hours, green fluorescent protein activity was measured (internal control for normalization) followed by the addition of Steadylite plus (PerkinElmer) to determine luciferase activity, both of which were measured in a PheraStar FS (BMG) plate reader. Transfection experiments were performed three times, and the results are shown as mean values of triplicates and standard deviations.
9
Plasmid Purification and Transfection Workflow
10
Measuring Peptide-Induced cAMP Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cotransfected HEK293T cells were detached, counted and pelleted as described above. Cells were resuspended at a concentration of 106 cells/ml in DMEM/F-12 supplemented with 200 μM IBMX for studying stimulatory effects or supplemented with 200 μM IBMX and 20 μM NKH-477 (H2O: soluble analogue of forskolin; Sigma-Aldrich) to study inhibitory effects. DMEM/F-12 supplemented with 200 μM IBMX was used as negative control, DMEM/F-12 containing 200 μM IBMX and 20 μM NKH-477 was used as positive control. Dilution series were made for the SchgrETH peptides. 50 μl of peptide was added to the wells of the 96-well plate already containing 50 μl of cells suspension. The plate was subsequently incubated for 3–4 h at 37 °C and 5% CO2. 100 μl of SteadyLite Plus (Perkin Elmer) was added to the wells of the plate, after which the plate was gently shaken at room temperature for 15 min in the dark. Luciferase activity in the shape of light emission was measured for 5 s/well using a Mithras LB940 (Berthold Technologies). Data were analysed as described above.
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!