The RH7777-hLPA5 cells were purchased from MultiSpan, Inc. and validated for activity in our laboratory using the known LPA5 agonist, hexadecyl LPA 16:0. Cell culture materials, assay consumables, and assay reagents were purchased from Fisher SSI. The Calcium 5 dye was purchased from Molecular Devices and the LanceUltra kit (TRF0262) was purchased from PerkinElmer.
Calcium 5 dye
Manufactured by Molecular Devices
Sourced in United States
Calcium 5 dye is a fluorescent indicator used for the detection and measurement of calcium ion (Ca2+) levels in biological samples. It binds to Ca2+ ions, leading to a change in its fluorescence properties that can be detected using fluorescence-based instruments. The dye is designed for use in a variety of cell-based assays and experiments involving the monitoring of intracellular calcium dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Flipr tetra, by Molecular Devices (2 mentions)
Screenworks 4, by Molecular Devices (2 mentions)
Hyclone hepes buffered saline, by GE Healthcare (2 mentions)
Biocoat, by Corning (2 mentions)
Lance ultra kit, by PerkinElmer (1 mentions)
Flipr, by Molecular Devices (1 mentions)
Calcium ionophore a23187, by Merck Group (1 mentions)
Flipr tetra plate reader, by Molecular Devices (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Probenecid, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Merck Group (1 mentions)
8 protocols using calcium 5 dye
1
LPA5 Receptor Activation in RH7777 Cells
2
Orexin-induced Calcium Response Assay
3
Calcium and Membrane Potential Assays in HEK-293 Cells
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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
Cannabinoid Receptor Activity Evaluation
5
High-throughput Calcium Flux Assay for α7 Nicotinic Receptors
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High-throughput FLIPR assays were conducted using 384-well BioCoat (Corning) plates. Cells were washed briefly in assay buffer (HyClone™ HEPES-buffered saline [GE Life Sciences] comprising 149 mM NaCl, 4 mM KCl, 10 mM HEPES, and 5 mM glucose at pH 7.4 and 300 mOsm osmolality, supplemented with 2 mM CaCl2 and 1 mM MgCl2) prior to Calcium5 dye (Molecular Devices) loading for 1 h at RT. Following removal of excess dye, plates were placed in the FLIPR Tetra (Molecular Devices) chamber, and fluorescent Ca2+ signal was captured using ScreenWorks 4.0™ software (Molecular Devices).
To obtain neuronal α7 mediated FLIPR responses, we used 5 μM PNU-12059615—a selective drug that attenuates receptor desensitization. Tetrodotoxin, or TTX (500 nM) was included in the assay buffer to inhibit spontaneous action potentials.
To obtain neuronal α7 mediated FLIPR responses, we used 5 μM PNU-12059615—a selective drug that attenuates receptor desensitization. Tetrodotoxin, or TTX (500 nM) was included in the assay buffer to inhibit spontaneous action potentials.
6
Quantifying TRPV4-mediated Calcium Influx
7
High-throughput FLIPR Assays for α7 nAChR
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High-throughput FLIPR assays were performed using 384-well BioCoat (Corning) plates. Assay buffer consisted of the HyClone™ HEPES-buffered saline (GE Life Sciences) described above, though prior to recordings, cells were loaded with Calcium 5 dye (Molecular Devices) for 1 h at room temperature. Plates were washed to remove excess dye, and then placed in the FLIPR Tetra (Molecular Devices) chamber. Recordings were captured using ScreenWorks 4.0™ software (Molecular Devices). Obtaining robust α7-mediated FLIPR responses required the presence of a selective PAM to attenuate desensitization, either 50 μM NS-1738 70 (link) for HEK293T cells or 5 μM PNU-120596 15 (link) for neurons. Neuronal FLIPR responses were also obtained in the presence of tetrodotoxin, or TTX (500 nM), to inhibit the firing of action potentials.
8
T-type Calcium Channel Screening Assay
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Calcium 5 dye (Molecular Devices, United States ) was used to investigate compound activities at T-type calcium channels using Flexstation III. HEK293 cells overexpressing Cav3.1, Cav 3.2, and Cav 3.3 cells were resuspended in Leibovitz’s L-15 media containing 1% FBS, 1% P/S and 15 mM glucose. 90 µl of the resuspended cells (120,000 cells/well) were plated in a black-walled, 96-well microplate (Corning, NY, United States ) and incubated overnight in humidified air at 37°C. Channel expression was induced with tetracycline (2 μg/ml, Sigma-Aldrich) at plating. On the day of the assay, 90 µl of HBSS containing 5% Calcium dye (bulk kit) and 2.5 mM probenecid (Invitrogen, United States ) was added to each well and incubated for 1 h at 37°C. Change in fluorescence was measured in a FlexStation III plate reader every 2 s (ʎexcitation = 485 nm, ʎemission = 525 nm) for the duration of the experiment at 37°C. After 2 min baseline recording, drugs (20 µL) were added and read for 5 min, followed by 10 mM CaCl2 (20 µl) and read for a further 3 min.
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