HK-2 cells were seeded into 384-well microplates with 8 × 103 cells per well and grown overnight. After exposure to U at 0, 100, and 600 μM for 24 h, the cells were incubated with 25 µL of the dye loading solution containing Fluo-4 AM and probenecid at 37 °C for 30 min, then at room temperature for an additional 30 min using Fluo-4 NW Calcium Assay Kit (Thermo Fisher Scientific, #F36206) according with the manufacturer instruction. Fluorescence intensity was recorded at excitation wavelength of 470 nm and emission wavelength of 516 nm by an FLIPR Penta high-throughput real-time fluorescence imaging analysis system (FLIPR) operated by FLIPR Penta (Molecular Devices). The changes of Fluo-4 fluorescence ΔF over basal fluorescence F0 (ΔF/F0) were used to monitor the changes of cytosolic [Ca2+] upon stimulation. Ionomycin (2 μM, Yeasen Biotechnology Shanghai, China, #50401ES03) was added at the beginning of all experiments to induce Ca2+ release from ER Ca2+ stores77 (link),78 (link). ML-SA1 at 10 μM was added after Ionomycin treatment to induce the Ca2+ release from lysosomes.
Flipr penta
Manufactured by Molecular Devices
Sourced in United States
The FLIPR Penta is a high-throughput screening system designed for cellular assays. It enables the simultaneous measurement of multiple fluorescent readouts from up to 1,536 wells in a microplate. The system is equipped with a high-sensitivity camera, dual-excitation light sources, and a temperature-controlled microplate chamber to provide reliable and reproducible data.
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Lab products found in correlation
Edelfosine, by Bio-Techne (1 mentions)
U73122, by Merck Group (1 mentions)
Xestospongin c, by Abcam (1 mentions)
Spectramax gemini em, by Molecular Devices (1 mentions)
Ionomycin, by Yeasen (1 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
Flipr, by Molecular Devices (1 mentions)
Screenworks, by Molecular Devices (1 mentions)
5 protocols using flipr penta
1
Calcium Signaling Regulation in HK-2 Cells
2
Ca2+ Imaging of Microglia Activation
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Mouse macrophages and microglia were washed with HBSS and then loaded with 2 µM Fluo8‐AM solution (Stratech) at room temperature in the dark for 1 h. Cells were then washed and imaged in Ca2+‐free media and EGTA used as a negative control. Plates were warmed in a plate reader (Spectramax Gemini EM) or automated cell screening system (FLIPR Penta Molecular Devices) to 37°C and cells were exposed at set time points to anti‐FcγRII/III, LPS, oligomers of Aβ1–42 or DOPS‐liposomes (25 μg/ml), as above. This was followed by ionomycin (2 µM) as a positive control. Levels of fluorescence were detected at ex 490 nm/ em 520 nm (Hagen et al, 2012 (link)). Cells were inhibited by pre‐exposure for 2 h with edelfosine (10 µM) (Tocris) or U73122 (5 µM) (Sigma) or xestospongin C (5 µM) (Abcam). Similarly, hiPSC‐derived microglia were exposed to anti‐CD32 (Fisher 16‐0329‐81, 5 μg/ml) or DOPS‐liposomes (25 μg/ml).
3
High-Throughput Fluorescent Imaging of Calcium Responses
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For high-throughput fluorescent imaging assays using the FLIPRPenta (Molecular Devices), RealDRG™ were thawed and cultured in Chrono™ Senso-MM Cells were seeded at a density of 1000–10,000 cells/well on black-walled 384-well imaging plates (Corning, cat# CLS3657) pre-coated with Matrix3 and cultured in Anatomic Chrono™ Senso-MM. To record fluorescence responses following stimulation with agonists, cells were loaded with Calcium 4 dye kit (Molecular Devices, cat# R8141) diluted according to the manufacturer’s instructions in physiological salt solution (PSS, composition in mM: NaCl 140, glucose 11.5, KCl 5.9, MgCl2 1.4, NaH2PO4 1.2, NaHCO3 5, CaCl2 1.8, HEPES 10) and incubated at 37 °C for 30 min. Responses were measured every 1 s for 300 s and, analysed using ScreenWorks 5.1.1.86 (Molecular Devices).
4
Functional Characterization of GPI Complex
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HEK293-NaV1.7 cells were transfected with TMEM233, CRELD1, LMAN2L, MMGT1, RNF121 and STT3B and co-transfected with five plasmids coding for the Glycosylphosphatidylinositol (GPI) complex (GPAA1, PIGK, PIGS, PIGU, PIGT) using Lipofectamine 2000. After 24 h, transfected cells were harvested and plated at a density of 10,000–15,000 cells per well on black-walled 384-well imaging plates. Plated cells were cultured for a further 24 h at 37 °C and 5% CO2. FLIPR (Fluorescent Imaging Plate Reader) Red membrane potential assay dye (Molecular Devices, California, USA) was diluted in physiological salt solution according to the manufacturer’s instructions (PSS, in mM: NaCl 140, glucose 11.5, HEPES 10, KCl 5.9, MgCl2 1.4, NaH2PO4 1.2, NaHCO3 5, CaCl2 1.8, pH 7.4). Growth medium was removed from the plate and replaced with 20 µL of FLIPR Red membrane potential assay dye per well and incubated in the dark at 37 °C for 60 min. Changes in membrane potential were assessed using a FLIPRPenta (excitation, 515–545 nm; emission, 565–625 nm; Molecular Devices, California, USA) every 0.5 s for 10 min after the addition of ExTxA (10 µM). Fluorescence responses to ExTxA were computed as the maximum change in fluorescence over baseline (ΔF/F0) using ScreenWorks (Molecular Devices, Version 5.1.2.94).
5
Measuring GPCR Activation via BRET
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HEK293A.CXCR2 cells were transiently co-transfected with plasmids encoding Gα protein tagged with NLuc (donor) and Gγ protein tagged with LSS-mKATE2 (acceptor) in a 1:10 donor-acceptor ratio [29 (link)]. Forty-eight hours after transfection and seeding, cells were washed with assay buffer and incubated with 90 μL of a 1:100 Nano-Glo® Vivazine™ working solution (#N2581, Promega) for 45 min at 37°C and 5% CO2. If navarixin was added, it was dissolved in the Vivazine solution at a concentration of 1 μM. The plate was transferred to the FLIPR Penta (Molecular Devices). After 15 min of equilibration time, baseline BRET signals were determined by five consecutive measurements immediately followed by the automatic addition of 10 μL of 10 × ligand to the cell plate by the FLIPR Penta. Changes in BRET ratios were monitored in real-time (every 2.5 s) for 25 min. Measurements were acquired using a 440-480 nm donor emission filter (#0200–6179, Molecular Devices) and a custom 615 nm AT600lp acceptor emission filter (#296420, Chroma).
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