CHO-NFAT cells stably expressing the human M 1 receptor, developed in-house, were plated in growth media at 3000 cells/well (8 µL of 3.75 × 105 cells/mL per well) into black, cyclic olefin copolymer (COC) tissue culture-treated, clearbottom, 1536-well plates (Corning) using a GNF bottle valve dispenser (GNF, San Diego, CA) and incubated overnight at 37 °C, 85% to 90% humidity, and 5% CO 2 (Liconic, Mauren, Liechtenstein). The assay was initiated 20 h later by washing the cells twice with assay buffer, leaving 2 µL of volume and dispensing 4 µL calcium dye using the GNF Washer (GNF). Plates were incubated for 50 min at 37 °C, 85% to 90% humidity, and 5% CO 2 . Following the incubation, the assay plate was read on a FLIPR-Tetra (Molecular Devices, Sunnyvale, CA) using excitation filters of 470 to 495 nm and emission filters of 515 to 575 nm for 5 s. The 1536-well elastomeric head of the FLIPR-Tetra was used to transfer 2 µL of the PAM compound plate into the assay plate during the read. The elastomeric head was washed and sonicated in 50/50 DMSO/H 2 O after each transfer and dried at a vacuum station within the FLIPR-Tetra. The plate was imaged for 70 additional seconds. The final reported data for each well were the maximum/minimum or the ratio of the maximum peak signal normalized for the baseline signal, which was averaged for 5 s before agonist addition.
Flipr tetra
Manufactured by Molecular Devices
Sourced in United States, United Kingdom
The FLIPR Tetra is a high-throughput cellular screening system designed for rapid and sensitive detection of cellular responses. It utilizes fluorescence-based detection to measure changes in cellular parameters, such as membrane potential and calcium flux, in real-time. The FLIPR Tetra is capable of simultaneously monitoring multiple wells in a microplate format, making it a versatile tool for a wide range of applications in drug discovery and cell biology research.
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Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (15 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Probenecid, by Merck Group (7 mentions)
Screenworks 4, by Molecular Devices (7 mentions)
Screenworks software, by Molecular Devices (5 mentions)
Earlytox cardiotoxicity kit, by Molecular Devices (4 mentions)
Pbx no wash ca2 assay kit, by BD (4 mentions)
Somatostatin 14, by Bachem (4 mentions)
Fluo 4 nw, by Thermo Fisher Scientific (4 mentions)
Fluo 8, by AAT Bioquest (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Calcium 4 dye, by Molecular Devices (3 mentions)
Cellbind plate, by Corning (3 mentions)
Calcium 6 dye, by Molecular Devices (2 mentions)
Hepes, by Merck Group (2 mentions)
Hbss buffer, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Red membrane potential dye, by Molecular Devices (2 mentions)
Probenecid, by Thermo Fisher Scientific (2 mentions)
Black poly d lysine coated 384 well plates, by Greiner (2 mentions)
104 protocols using flipr tetra
1
Calcium Signaling Assay for M1 Receptor
2
Phenotyping and Imaging of Cellular Models
3
Calcium Uptake in Cardiac SR Vesicles
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Calcium uptake by pig cardiac SR vesicles was measured by adding 30 µg/mL of sample to a buffer containing 50 mM MOPS (pH 7.0), 100 mM KCl, 30 mg/mL sucrose, 1 mM EGTA, 10 mM KOA, 2 µM Fluo-4, and various amounts of CaCl2 calculated to reach the desired free [Ca2+]. The assay mix was dispensed into pre-plated 384-well drug plates and incubated at room temperature for 20 min while protected from light. A final concentration of 5 mM MgATP was added to start the reaction and the decrease in the fluorescence excited at 485 nm was monitored at 520 nm for 15 min using a FLIPR Tetra (Molecular Devices, San Jose, CA, USA).
4
Evaluating Antibody Inhibition of CXCL12-induced Calcium Flux in Jurkat Cells
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The ability of PF-06747143 or m15-IgG1 to inhibit CXCL12-induced calcium flux was evaluated in human T cell leukemia Jurkat cells using the Fluo-NW Calcium assay kit (Life Technologies). Cells were plated in 384-well plates at 70,000 cells per well in quadruplicates and incubated with m15-IgG1 parent antibody and PF-06747143, upon stimulation with CXCL12 at 8 nM (EC80) (Invitrogen), for 110 min. Calcium flux was then measured for 95 s using a FLIPR Tetra (Molecular Devices).
5
Ca2+ Transport Assay of SERCA2a
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Ca2+-transport assays were performed with similar porcine SR samples as in the Ca2+-ATPase assays described above. The compound effect on the Ca2+-transport activity of SERCA2a was determined using an oxalate-supported assay in which the change in fluorescence in a Ca-sensitive dye, Fluor-4, was determined as previously described18 (link). A buffered solution containing 50 mM MOPS (pH 7.0), 100 mM KCl, 30 mg/mL sucrose, 1 mM EGTA, 10 mM potassium oxalate, 2 μM Fluo-4, 30 μg/mL porcine cardiac SR vesicles, CaCl2 calculated to reach the free [Ca2+] (pCa 8.0, 6.2, and 5.4), and compound (0.048 to 50μM) was dispensed into 384-well black walled, transparent bottomed plates (Greiner Bio-One) containing the tested small molecule and incubated at 22°C for 20 minutes while covered and protected from light. To start the reaction, MgATP was added to a final concentration of 5 mM, and the decrease in 485-nm excited fluorescence of Fluo-4 was monitored at 520 nm for 15 min using a FLIPR Tetra (Molecular Devices, San Jose, CA).
6
Screening Mutant Library with FLIPR Tetra
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384-well assay plates were washed with HANKS buffer, loaded with fluo-3 and washed with HANKS buffer as described before [25] (link). The mutant library was screened on a FLIPR Tetra (Molecular Devices) in two independent experiments: First, 100 µM MO followed by 1 µM Compound 31 (final concentrations) was added. Prior to usage DMSO stock solutions of MO and Compound 31 were diluted in HANKS buffer. Second, 384-well assay plates were cooled from 25°C to 10°C using a custom-made device [25] (link).
7
GABAA Receptor Potentiation Screening
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A membrane potential FLIPR assay was developed and validated using a Molecular Devices FLIPR TETRA in 384‐well plate format. Single‐point screening (10 μmol·L–1) of a repurposing library of 1320 compounds identified activators and potentiators of the WT GABRA3/GABRB3/GABRG2 GABAA receptor using the clonal HEK293 cell line expressing this receptor. A voltage‐sensitive fluorescent dye (R8042, Molecular Devices) was loaded into cultured cells. Cells were washed and then incubated in test compound for 4 minutes followed by addition of GABA to a concentration of 15 nmol·L–1 (near the EC20 for GABA stimulation for this assay). Percentage potentiation was measured and compared to the maximum response (15 nmol·L–1 GABA and 1 μmol·L–1 diazepam). Potency determination with eight‐point CRCs in half‐log dilution steps starting at 30 μmol·L–1 was performed for selected compounds. The EC50 of reference compounds diazepam and phenobarbital in this assay was 28 nmol·L–1 and 90 μmol·L–1, respectively.
8
Calcium Oscillation Measurement Assay
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Medium was removed and cells were incubated in assay buffer (HBSS, Gibco, 10 mM HEPES, pH 7.4) containing 4 µM Fluo-4AM (Thermo Fischer Scientific) and 0.1% Pluronic F-127 (Thermo Fischer Scientific #P3000MP) for 1 h at room temperature in the dark. Loading buffer was then replaced with assay buffer. After 10 min, assay plates were measured in the FLIPR tetra (Molecular Devices, California, USA) using green fluorescence filter settings (excitation 470–495 nm, emission 515–575 nm). Fluorescence signals were recorded at 1 s intervals (0.4 s exposure time). Compound addition was automated in a single addition protocol. Tested compounds (concentrations:0.3 nM–1 µM; retigabine at 30 µM) were added after recording a baseline response for 5 min followed by another 5 min for recording compound effects on Ca2+ oscillations. Each compound dose was repeated on three individual plates in duplicates.
9
Intracellular Calcium Flux in iPSC Cardiomyocytes
10
Calcium Signaling Modulation in Neuroblastoma Cells
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The assays were performed using a fluorescence imaging plate reader (FLIPR TETRA; Molecular Devices®) with the neuroblastoma cell line SH-SY5Y. SH-SY5Y endogenously expresses the voltage-gated channels of sodium (Nav 1.2, 1.3, and 1.7), calcium (L and N-type), and ligand-gated nAChR channels (α3- and α7-containing)35 (link),36 (link). SH-SY5Y cells were cultured in RPMI containing 15% fetal bovine serum (FBS) and supplemented with L-glutamine. Cells were grown to 70–80%, plated at a density of 50,000 cells/well on black-walled 384-well imaging plates, and cultured in a humidified incubator set at 37 °C and 5% CO2 for 48 h. Next, the medium was removed, and SH- SY5Y cells were loaded with 20 µl/well Calcium 4 dye reconstituted in assay buffer 36 (link). The fluorescence was read every second and expressed as a response over baseline. The peptide (100–0.024 µM), protonectin or protonectin-F, was added after 10 baseline reads. Then, after 5 min, cells were stimulated by the addition of Veratridine (50 µM) or Nicotine (30 µM), and fluorescence was recorded for a further 5 min.
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