Membranes were prepared from CHO‐K1 cells stably or transiently expressing recombinant wild‐type (wt) human smoothened receptors (SMO) or transiently expressing mutant SMO M2 (SMO W535L) or SMO D473H variants. Receptor‐mediated G‐protein activation was determined by measuring [35S]GTPγS (1250 Ci/mmole; NEN, PerkinElmer, France) incorporation. Briefly, membranes were preincubated 30 min at 30°C with receptor ligands in a buffer containing 20 mM HEPES, pH 7.4, 3 μM GDP, 3 mM MgCl2, 100 mM NaCl. The reaction was started by the addition of 0.5 nM [35S]GTPγS in a final volume of 500 μL in 96‐well plates and incubation was performed for an additional 30 min. Experiments were terminated by rapid filtration through Unifilter‐96 GF/B filters (PerkinElmer Life Sciences, Boston, MA, USA) using a Filtermate harvester (PerkinElmer Life Sciences). Radioactivity retained on the filters was determined by liquid scintillation counting using a TopCount microplate counter (PerkinElmer Life Sciences). Inhibition of constitutive receptor activity was determined relative to the effect induced by the reference antagonist, cyclopamine. Basal binding was therefore defined as 0 %, whereas the effect of cyclopamine (10 μM) on basal [35S]GTPγS incorporation was defined as full inverse agonism (−100%).
Topcount microplate counter
Manufactured by PerkinElmer
The TopCount microplate counter is a precision instrument designed for high-throughput measurement of radioactivity or luminescence in microplates. It offers accurate and reliable detection capabilities for a wide range of research and screening applications.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using topcount microplate counter
1
Measuring Smoothened Receptor Activation
2
Cytotoxicity Evaluation of Ovarian Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The determination of in vitro drug cytotoxicity was assessed by the ATPlite luminescence adenosine triphosphate (ATP) detection assay system (PerkinElmer, Zaventem, Belgium), according to the manufacturer's instructions. All ovarian cancer cell lines were resuspended in complete medium and seeded into 96-well flat-bottomed plates (8 × 103/well). The day after, different concentrations of drugs were added (final volume, 100 μL/well) for 72 hours. At day 4, 50 μL of lysis solution were added to each well followed by addition of 50 μL of substrate solution. Finally, the luminescence was counted by the TopCount Microplate Counter (PerkinElmer). Within each experiment, determinations were performed in triplicate and experiments were repeated 5 times for each cell line. The percentage of cell survival was calculated by determining the counts per second (cps) values according to the formula: [(cpstested − cpsblank) / (cpsuntreated control − cps blank)] × with 100, with cpsblank referring to the cps of wells that contained only medium and ATPlite solution. The IC50 values were calculated from semi-logarithmic dose-response curves by linear interpolation.
3
In vivo Luciferase Assay Protocol
4
In vitro Cytotoxicity Assay of Drugs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro cytotoxicity of free drugs, ONCOFID-P, ONCOFID-S and fluorochrome-labeled bioconjugates was assessed against all cell lines using the ATPlite luminescence adenosine triphosphate (ATP) detection assay system (PerkinElmer, Zaventem, Belgium) [41] (link), according to the manufacturer's instructions. Briefly, cells were resuspended in complete medium and seeded into 96-well flat-bottomed plates (8×103/well); the day after, different drug concentrations were added (final volume, 100 µL/well) for 72 hours. At day 4, 50 µL of lysis solution were added to each well followed by addition of 50 µL of substrate solution and final counting of luminescence by the TopCount Microplate Counter (PerkinElmer). Within each experiment, determinations were performed in triplicate and experiments were repeated 5 times for each cell line. The percentage of cell survival was calculated by determining the counts per second (cps) values according to the formula: [(cpstested - cpsblank)/(cpsuntreated control - cpsblank)]×100, with cpsblank referring to the cps of wells that contained only medium and ATPlite solution. IC50 values were calculated from semi-logarithmic dose-response curves by linear interpolation.
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!