Calcium flux in cardiomyocytes was assessed using the instructions provided by the EarlyTox Cardiotoxicity Kit from Molecular Devices, LLC (Table 1 ). Briefly, cells in 25 μL medium per well were equilibrated for 2 h at 37°C in the presence of 25 μL of prewarmed calcium dye reagent. Before treatment with test chemicals, basal calcium flux was recorded at 515–575 nm following excitation at 470–495 nm for 100 s in 0.125 s read intervals using the FLIPR tetra plate reader (Molecular Devices). The exposure time per read was 0.05 s, the gain was set to 2,000, and the excitation intensity was set to 30%. The instrument temperature was kept at a constant 37°C. Test chemicals (12.5 μL of 5× concentration working solutions) at the appropriate concentrations were added simultaneously to all wells using the instrument-specific automated liquid handler. Between subsequent readings at 10, 30, 60, and 90 min, cells were incubated at 37°C and 5% CO2.
Flipr tetra plate reader
Manufactured by Molecular Devices
Sourced in United States
The FLIPR TETRA plate reader is a high-throughput screening instrument designed for cellular assays. It is capable of simultaneously measuring fluorescent signals from multiple wells in a microplate. The device is equipped with a high-intensity light source, sensitive detectors, and advanced software to enable rapid data acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Echo 550, by Beckman Coulter (2 mentions)
Earlytox cardiotoxicity kit, by Molecular Devices (1 mentions)
384 well plate, by Greiner (1 mentions)
Multidrop dispenser, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Calcium ionophore a23187, by Merck Group (1 mentions)
Calcium 5 dye, by Molecular Devices (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Neurotransmitter transporter uptake assay kit, by Molecular Devices (1 mentions)
5 protocols using flipr tetra plate reader
1
Calcium Flux Assay in Cardiomyocytes
2
High-throughput Screening for USP30 Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
USP30 inhibitors were dispensed into black, clear bottom, low binding 384 well plates (Greiner) using the ECHO 550 (Labcyte) liquid handler. An amount of 75 nl of compound was dispensed in 100% DMSO. Total reaction volume was 30 µl producing an USP30 inhibitor top concentration of 25 µM. His-tagged, 2× concentrated recombinant human USP30 protein (rhUSP30; amino acids 57–517 of the full-length protein, and a C-terminal 6-His tag, Sf 21 (baculovirus)-derived human USP30 protein) (10 nM; final assay concentration = 5 nM; Boston Biochem) was prepared in USP30 activity assay buffer (50 mM Tris base pH 7.5, 100 mM NaCl, 0.1 mg/ml BSA (Sigma, A7030), 0.05% Tween 20, 1 mM DTT) and 15 µl dispensed into compound containing assay plate using the Multidrop dispenser (Thermo Scientific) and incubated for 30 min at room temperature. Following incubation, 15 µl of 2× concentrated ubiquitin-rhodamine 110 (Ub-Rho110) (200 nM, final assay concentration = 100 nM; Boston Biochem), prepared in USP30 activity assay buffer, was dispensed into the compound-rhUSP30 containing plate and fluorescence immediately read on the FLIPR TETRA plate reader (Molecular Devices). Fluorescence was recorded every 30 s over 1 h and intensity was analysed.
3
Calcium and Membrane Potential Assays in HEK-293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293 cells
(ATCC CRL-1573) were seeded at 15000 cells/well onto poly(d -lysine) coated black/clear bottom 384-well plates in 25 μL
of minimum essential medium, 10% FBS, 100 IU/mL penicillin, 100 μg/mL
streptomycin, and 2 mM Glutamax. Compounds were diluted in DMSO and
tested as 10-point, 3-fold serially diluted samples starting at 100
μM. For the calcium ionophore assay, 25 μL of calcium
5 dye (Molecular Devices) was suspended in Hank’s Balanced
Salt Solution (HBSS) with 20 mM HEPES, pH 7.4, and added to 384-well
cell plates containing HEK-293 cells, then incubated for 60 min at
37 °C and 5% CO2. Compounds were then added to the
dye-loaded cell plates. Using a FLIPR Tetra plate reader (Molecular
Devices), fluorescent signal (excitation 470–490 nm; emission
515–575 nm) was detected at 1 s intervals for 300 s. Emission
maximum results, a measure of intracellular calcium, were plotted
using XLfit software (IDBS, Inc.). Calcium ionophore A23187 (Sigma)
was used as a positive control. The membrane potential assay was carried
out as described above for the calcium ionophore assay except membrane
potential red dye R8126 (Molecular Devices) was used and detected
at excitation 510 nm and emission 565–625 nm to indicate voltage
depolarization changes across the cell. Amphotericin B (Sigma) was
used as a positive control.
(ATCC CRL-1573) were seeded at 15000 cells/well onto poly(
of minimum essential medium, 10% FBS, 100 IU/mL penicillin, 100 μg/mL
streptomycin, and 2 mM Glutamax. Compounds were diluted in DMSO and
tested as 10-point, 3-fold serially diluted samples starting at 100
μM. For the calcium ionophore assay, 25 μL of calcium
5 dye (Molecular Devices) was suspended in Hank’s Balanced
Salt Solution (HBSS) with 20 mM HEPES, pH 7.4, and added to 384-well
cell plates containing HEK-293 cells, then incubated for 60 min at
37 °C and 5% CO2. Compounds were then added to the
dye-loaded cell plates. Using a FLIPR Tetra plate reader (Molecular
Devices), fluorescent signal (excitation 470–490 nm; emission
515–575 nm) was detected at 1 s intervals for 300 s. Emission
maximum results, a measure of intracellular calcium, were plotted
using XLfit software (IDBS, Inc.). Calcium ionophore A23187 (Sigma)
was used as a positive control. The membrane potential assay was carried
out as described above for the calcium ionophore assay except membrane
potential red dye R8126 (Molecular Devices) was used and detected
at excitation 510 nm and emission 565–625 nm to indicate voltage
depolarization changes across the cell. Amphotericin B (Sigma) was
used as a positive control.
4
Neurotransmitter Transporter Uptake Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescent substrate uptake assays were performed using the Neurotransmitter Transporter Uptake Assay Kit (Molecular Devices, San Jose, CA, USA) following the supplier’s protocol. JumpIn-NET cells were seeded at 20,000 cells/well in culture medium in presence of 1 μg/ml dox in clear-bottom, black-walled 384 microtiter plates pre-coated with poly-D-lysine (Twin Helix, Milan, Italy) at 37 °C and 5% CO2. After 24 h medium was removed and 20 μl/well of Standard Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM NaHCO3, 20 mM HEPES, pH 7.4) was added. Cells were pretreated by addition of 10 μl/well NET inhibitor (increasing concentrations), inhibitor control (30 μM desipramine) or vehicle control (buffer only) in Standard Tyrode’s buffer at 0.1% DMSO (final concentration) for 1 h. Subsequently, cells were treated with 15 μl/well loading dye solution in Standard Tyrode’s buffer. Cells were incubated for 1 h, after which the fluorescence was measured for 60 s using a FLIPRTETRA plate reader (Molecular Devices, San Jose, CA, USA). All conditions were tested in quadruplicate on each plate except the vehicle and inhibitor controls, which each had 16 replicates per plate.
5
Fluorometric Assay for USP30 Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
USP30-UbRho10 was performed as previously described [39] (link) . Compounds were dispensed into black, clear-bottom, 384-well plates (Greiner, 781096) using the ECHO 550 (Labcyte) liquid handler. 2 × FAC His-tagged recombinant human USP30 protein (rhUSP30; amino acids 57-517 of the full-length protein, and a C-terminal 6-His tag, Sf 21 (baculovirus)-derived; 10 nM, FAC of 5 nM [R&D Systems, E-582-050]) was prepared in USP30 activity assay buffer (50 mM Tris base pH 7.5, 100 mM NaCl, 0.1 mg/mL BSA [Sigma, A7030], 0.05% Tween 20, 1 mM DTT) and 15 μL dispensed into compound-containing assay plate using the Dragonfly Discovery liquid dispenser and incubated for 30 min at room temperature. Following incubation, 15 μL of 2x concentrated ubiquitin-rhodamine 110 (Ub-Rho110; 200 nM, FAC of 100 nM [R&D Systems, U-555-050]) in USP30 activity assay buffer was dispensed into the compound-rhUSP30 containing plate using the Dragonfly Discovery liquid dispenser and fluorescence read on the FLIPR TETRA plate reader (Molecular Devices). Fluorescence intensity was recorded over 1 h and normalized to positive and negative controls (USP30Inh-1 [39] (link) , 10 μM and DMSO respectively).
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!