After three days of culture, the change in ATP release and viability of E12.5 vmDA neuron in response to gabapentin treatment were measured by alamarBlueTM Cell Viability Reagent (Thermo Fisher, USA) according to the manufacturer’s guidelines. The fluorescence was measured using a PHERAstar FS (BMG LabTech, Germany). The CellTiter-Glo® 3D Cell Viability Assay (Promega, G9681) was used to determine the status of the metabolic activity and ATP release of the cells. The luminescent signal was read by PHERAstar FS plate reader (BMG LabTech, Germany).
Pherastar fs
Manufactured by BMG Labtech
Sourced in Germany, United Kingdom, United States, Denmark, France
The PHERAstar FS is a high-performance microplate reader designed for a wide range of applications in life science research. It is capable of fluorescence, luminescence, and absorbance measurements, making it a versatile instrument for various assay types. The PHERAstar FS utilizes a multi-mode detection system to provide accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Mars data analysis v 3.01r2, by BMG Labtech (15 mentions)
H2dcfda, by Thermo Fisher Scientific (6 mentions)
Prism 8, by GraphPad (4 mentions)
Multidrop combi reagent dispenser, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Dcf da, by BMG Labtech (3 mentions)
Lps from e coli serotype 055 b5, by Merck Group (3 mentions)
Prism 6, by GraphPad (3 mentions)
Prism 7, by GraphPad (2 mentions)
Celltiter glo reagent, by Promega (2 mentions)
Pherastar mars, by BMG Labtech (2 mentions)
Clariostar, by BMG Labtech (2 mentions)
2 7 dichlorodihydrofluorescein diacetate solution, by Thermo Fisher Scientific (2 mentions)
Infinite 500, by Tecan (2 mentions)
Rubystar, by BMG Labtech (2 mentions)
Enzcheck phosphate assay kit, by Thermo Fisher Scientific (2 mentions)
Multidrop combi, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Celltiter glo, by Promega (2 mentions)
Fluo 4 direct calcium assay kit, by Thermo Fisher Scientific (2 mentions)
237 protocols using pherastar fs
1
Gabapentin's Effects on Neuronal ATP and Viability
2
Ligand Binding Assay Using HTRF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Signal was detected using a PHERAstar FS (BMG Lab technologies, Offenburg, Germany) microplate reader equipped with a FRET optic module allowing donor excitation at 337 nm and signal collection at both 665 and 620 nm. A frequency of 10 flashes/well was selected for the xenon flash lamp excitation. The signal was collected at both 665 and 620 nm using the following time-resolved settings: delay, 150 ms; integration time, 500 ms. HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm) and multiplying this value by 10,000. The 10,000-multiplying factor is used solely for the purpose of easier data handling.
Data were analyzed using Prism 7 software (GraphPad Software, Inc., San Diego, CA), and competition data were fitted by non-linear regression to a one site-fit logIC50, competition curves were -in all cases- monophasic. KD (dissociation constant) values of the fluorescent ligand were obtained from the specific binding saturation curves. Note that Bmax values obtained from HTRF saturation curves do not reflect absolute values of receptor binding sites. Ki values were determined from competition binding assays by using the calculated IC50 and KD values and the Cheng-Prusoff equation [25] (link).
Data were analyzed using Prism 7 software (GraphPad Software, Inc., San Diego, CA), and competition data were fitted by non-linear regression to a one site-fit logIC50, competition curves were -in all cases- monophasic. KD (dissociation constant) values of the fluorescent ligand were obtained from the specific binding saturation curves. Note that Bmax values obtained from HTRF saturation curves do not reflect absolute values of receptor binding sites. Ki values were determined from competition binding assays by using the calculated IC50 and KD values and the Cheng-Prusoff equation [25] (link).
3
Carbachol-Induced IP-1 Accumulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells expressing receptors tagged with Cerulean and eYFP were either induced for 24 hours with 10 ng.mL -1 of doxycycline or left untreated. Cells were harvested using Versene dissociation reagent, washed, resuspended in Hank's Buffered Salt Solution (HBSS) and subsequently added to 384-well low volume Optiplates to a final cell density of 9800 cells/well. Cells were stimulated with variable concentrations of carbachol for 30 min at 37 o C. Detection of IP-1 accumulation was carried out for 1 hour at room temperature and protected form the light, using the IP-One Tb kit (Cisbio Bioassays, Bagnols-sur-Cèze, France) according to the manufacture's instructions. Plates were read using a PheraStar FS (BMG Labtechnologies, Offenburg, Germany).
4
Measuring Intracellular Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell suspensions (1 × 103 cells/well) were incubated during 1 h with 1 and 10 µM solutions of the tested compounds. Non-treated and treated with 6-OHDA at concentration of 50 µM (Sigma-Aldrich, USA) cells were used as negative and positive controls, respectively. The portion (20 µL) of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) stock solution (Molecular Probes, Eugene, OR, USA) with concentration of 100 mM was added in each well and the microplate was incubated for an additional 10 min at 37 °C. The intensity of dichlorofluorescin fluorescence was measured at λex = 485 nm, and λem = 518 nm with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany). The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). In other experiments, cells were incubated with compounds during 1 h. Then, 6-OHDA (50 µM) was added in each well for 30 min and ROS levels were measured. All obtained results were presented as percent of negative control data.
5
Mitochondrial ATP Synthesis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondria (1 mg protein ml−1 final concentration) were re-suspended in ATP synthesis buffer (20 mM Tris base 0.6 M sorbitol, 15 mM potassium phosphate monobasic, 10 mM magnesium sulphate, 2.5 mg ml−1 essentially fatty acid free BSA, final pH 7.4) containing either 10 mM L-glutamic acid, monosodium salt; 2 mM L-malic acid sodium salt or 10 mM succinate disodium salt; 1 μM rotenone and mitochondrial suspension (20 μl) was dispensed into a clear bottom black-walled 384 well plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Rockford, IL). Mitochondria were incubated in the presence of compound for 10 minutes. Assay buffer (20 μl) containing ADP (5 mM final assay concentration) was added to all wells and incubated at room temperature for 45 mins. The reaction was stopped by the addition of CellTiter-Glo reagent (Promega, Madison, USA) and the plate was left at room temperature for 10 minutes to stabilise. Luminescence was recorded using Pherastar FS (BMG Labtech, Ortenberg, Germany). Data were analysed using luminescent intensities, normalised to antimycin A (2.5 μM) in combination with oligomycin A (2.5 μg ml−1; 100% inhibition) and DMSO (0% inhibition).
6
Quantitative Luminescence Assay for NFAT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NFAT-RE-luc2 Jurkat cells were collected and centrifuged at 300 g and re-suspended in serum-free media containing supplements without antibiotics. Cell suspension (15,000 cells/20 μl) was added to each well of a solid black 384 well plate and incubated in the presence of compound for 45 minutes at 37 °C. A 3x solution containing ionomycin (final assay concentration 1 μM) and phorbol 12-myristate (PMA; final assay concentration 10 nM) was added to each well and cells incubated for 20 hours at 37 °C. An equal volume of Bright Glo reagent (Promega, Madison, WI) was added to all wells and plate allowed to stabilise for 10 minutes at room temperature. Luminescence was measured using the Pherastar FS (BMG Labtech, Ortenberg, Germany) and data normalised to CsA (5 μM; 100% inhibition) and DMSO (0% inhibition).
7
Mitochondrial Membrane Potential Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rat mitochondria (1 mg protein ml−1 final concentration) were incubated in assay buffer (75 mM mannitol, 25 mM sucrose, 5 mM potassium phosphate monobasic, 20 mM Tris base, 100 mM potassium chloride, 0.1% essentially fatty acid free BSA, adjusted to pH 7.4). The buffer was supplemented with either 10 mM L-glutamic acid, monosodium salt; 2 mM L-malic acid sodium salt as electron donor to complex I or 10 mM succinate disodium salt in combination with 1 μM rotenone as electron donor to complex II. Mitochondria were loaded with tetramethylrhodamine methyl ester (TMRM) (2 μM) for 10 minutes at room temperature and mitochondrial suspension (40 μl) was dispensed into each well of a solid bottom black 384 well plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Rockford, IL). Increased fluorescence intensity (ex. 540/em. 590 nm) indicated de-quenching of TMRM fluorescence and therefore mitochondrial membrane depolarisation40 (link). Fluorescence intensity was determined using Pherastar FS (BMG Labtech, Ortenberg, Germany) following 10 minutes compound incubation. Data were analysed using fluorescence intensities, normalised to antimycin A (2.5 μM) oligomycin A (2.5 μg ml−1; 100% depolarised) and DMSO (0% depolarised).
8
NFAT-RE-luc2 Jurkat Cell Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NFAT-RE-luc2 Jurkat cells were collected, centrifuged at 300 g for 5 minutes and re-suspended in serum-free RPMI-1640 media, plus supplements without antibiotic. Cell suspension (15,000 cells/20 μl) was added to each well of a solid black 384 well plate and incubated in the presence of compound for 20 hours at 37 °C. Alamar Blue was used according to manufacturer’s instructions and was added to each well at 2x concentration in media and reaction allowed to proceed for 4 hours at 37 °C. Fluorescence (ex. 540 nm/em. 590 nm) was recorded using Pherastar FS (BMG Labtech, Ortenberg, Germany).
9
CypD Binding Assay using HTRF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A CypD homogeneous time resolved fluorescence (HTRF) binding assay was developed to assess the binding affinities of compounds of interest (Cisbio, Codolet, France). Functionalised CsA-NH2 was tagged with the d2-acceptor molecule. Binding of CsA-d2 to his-tagged CypD (Anti-His-Lumi4®-Tb cryptate donor) was measured through assessment of TR-FRET intensity between acceptor (CsA-d2) and donor molecules (Anti-His- Lumi4®-Tb). The following volumes of working stocks were added to a solid black 384 well plate containing 100 nl compound/DMSO: 10 μl/well cyclophilin D, 5 μl/well CsA-d2, 5 μl/well Anti-His- Lumi4®-Tb cryptate. Reagents were incubated for approximately 3 hours before fluorescence intensity (ex. 620 nm/em. 665 nm) was measured using a PheraStar FS (BMG Labtech, Ortenberg, Germany). Data were normalised to CsA (2 μM; 100% binding) and DMSO (0% binding).
10
In Vitro PPIase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PPIase activity was measured in vitro using rhCypD and enzyme linked-confirmation specific cleavage of N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide to yield a colorimetric product. Ice-cold chymotrypsin (50 μl of 12 mg ml−1 in 1 mM hydrochloric acid) was added to each well of a clear non-binding 96 well plate containing compound. His-tagged-rhCypD (50 nM in 35 mM HEPES pH 7.9) was added in equal volume to each well of the compound plate and incubated on ice for 30 minutes. Substrate (4.5 μl of N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanalide; 5 mM in 450 mM lithium chloride) was dispensed in to a non-binding clear 96 well assay plate. The reaction was started through the addition of 90 μl of the chymotrypsin-rhCypD-compound solution to the assay plate and absorbance read at 405 nm using a Pherastar FS (BMG Labtech, Ortenberg, Germany). Data were collected at 3 second intervals over 60 seconds and area under the curve calculated. Data were normalised to CsA (2 μM; 100% inhibition) and DMSO (0% inhibition).
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!