Calcium currents were measured on crude synaptosomes. Briefly, after reactivation step, synaptosomes were pelleted as described before, resuspended in 300 μl 1 : 1 mixture using KRH and calcium sensitive cell-permeating fluorophore Fluo-4 (Fluo-4 Direct™ Calcium Assay, Life Technologies). Samples were placed in a whole black 96-well (Corning Costar® 96-Well Black Polystyrene Plate) for 25 min 37°C 5% CO2 to allow Fluo-4 to permeate cell membranes. After habituation fluorescence was measured with a Tecan F500 spectrophotometer for 180 cycles (corresponding to approximately 7 minutes) at 37°C. NMDA (100 μM, with glycine coagonist 1 μM final concentration) or KCl (50 mM final concentration) were injected 100 μl/sec at cycle 20 (45 sec). Fluorescence was recorded by a top mode reading optimizing Z-position.
Fluo 4 direct calcium assay
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Fluo-4 Direct™ Calcium Assay is a fluorescent dye-based assay designed to detect and measure intracellular calcium concentrations in live cells. It provides a sensitive and direct method for monitoring calcium-dependent cellular processes.
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Lab products found in correlation
Bzatp, by Merck Group (3 mentions)
Ionomycin, by Merck Group (2 mentions)
Costar 96 well black polystyrene plate, by Corning (1 mentions)
Fluo 4, by Corning (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Black clear bottom 96 well plates, by Corning (1 mentions)
Prism 6, by GraphPad (1 mentions)
Bay k8644, by Merck Group (1 mentions)
Fluo 4 direct calcium reagent, by Thermo Fisher Scientific (1 mentions)
Synergy 4 instrument, by Agilent Technologies (1 mentions)
Victor3 multilabel reader, by PerkinElmer (1 mentions)
Fluo 4 direct, by Merck Group (1 mentions)
Synergy ht biotek reader, by Agilent Technologies (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
10 protocols using fluo 4 direct calcium assay
1
Calcium Dynamics in Synaptosomes
2
Calcium Dynamics in NGF-SPIO-Au NPs Treated PC-12 Cells
3
Intracellular Calcium Release Assay in RAW264.7 Cells
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RAW264.7 cells were seeded in black, clear-bottom 96-well plates (Costar) at 20,000 cells per well. The next day, release of intracellular Ca2+ was measured using Fluo-4 (Fluo-4 Direct Calcium Assay; Life Technologies) according to the manufacturer’s instructions. Medium was discarded and 100 µL of 1 X Fluo-4 dye was incubated at 37 °C for 1 h in 5% CO2, protected from light. Then, basal fluorescence was measured using a fluorospectrometer (FLUOstar Omega; BMG Labtech), excitation at 485 nm and emission at 520 nm. Molecules of interest were then added with an electronic pipette; thapsigargin (10 µM, Sigma) as a positive control, 3-oxo-C12:2-HSL (1000 µM) or DMSO 0.5%; with 8 replicates per conditions for every experiment. The fluorescence signal was immediately measured every 80 s for 2 h. Results are expressed as a ratio of the fluorescence x times over the basal fluorescence (F/F0).
4
Fluo-4 Direct Calcium Assay in HL-1 Cells
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The Fluo-4 Direct Calcium Assay was performed as described by the manufacturer (Thermo Fisher Scientific). Briefly, HL-1 cells, pretreated as described, were stimulated with Fluo-4 Direct calcium reagent (Thermo Fisher Scientific) and signals were detected one hour post treatment. 10 mM Bay K8644 (Sigma) was added to cells and signals were immediately detected using a Synergy 4 instrument (BioTek). Results were analyzed using Prism 6.0 software (GraphPad Software, CA).
5
Measuring Cytosolic Calcium Dynamics under Hypoxia
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Cytosolic Ca2+ levels were measured using the “Fluo-4 Direct Calcium Assay” (ThermoFisher Scientific). Cells were seeded in black 96-well plates (3×104 per well) and incubated for 18 h under normoxic or hypoxic conditions. Subsequently, cells were challenged with TRAIL (256 ng/mL) for the indicated periods of time followed by direct addition of the Fluo-4-containing staining solution into the culture media. Fluorescence intensity was measured after 1 h incubation (37°C, protected from light) using a Victor3 multilabel reader (Perkin Elmer).
6
Intracellular Calcium Levels Quantification
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To measure intracellular calcium levels, Fluo-4 Direct™ Calcium Assay (Thermo Fischer Scientific) was performed following the manufacturer’s protocols. Briefly, 50 μl of the 2× Fluo-4 Direct™ calcium reagent loading solution was added to each well of a 96-well plate containing 50 μl of growth medium. The plate was then incubated at 37 °C for 30 min, followed by an additional 30 min incubation period at room temperature. Fluorescent signals were then measured using a fluorescence microscope. The intensity of the results was analyzed with the Image J program for three independent experiments.
7
Intracellular Calcium Dynamics Assay
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Changes in intracellular calcium concentrations were measured using Fluo-4 Direct™ Calcium Assay (ThermoFisher Scientific, UK) in accordance with the manufacturer’s instructions. In brief, 15,000 cells were loaded with Fluo-4 Direct™ calcium reagent, left to incubate for 1 h at 37 °C before being stimulated with 100 µM BzATP (Sigma, UK) (based on Agrawal et al 2020, [34] (link)) and then 0.8 µM ionomycin (Sigma, UK). Excitation ratio and emission wavelengths were 490 nm and 525 nm respectively.
8
Calcium Flux Analysis of Lanthipeptides
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Calcium efflux was measured by using a Fluo-4 Direct Calcium Assay purchased from Thermo Fisher Scientific. HEK293 cells with stable overexpression of GAL2R (HEKR2) (kindly provided by Harald Dargatz, Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, 53,115 Bonn, Germany) were used for analysis of the lanthipeptides. HEKR2 cells were seeded in a 96-well plate and grown overnight in DMEM supplemented with 10% FBS, 1% penicillin–streptomycin, 100 µg/ml Normocin, and 500 µg/ml G418 at 37 °C, 5% CO2 in a humidified incubator.
The next day, when the cells were nearly confluent, an equal amount of 2 × Fluo-4 Direct™ calcium reagent loading solution, containing 5 mM probenecid, was added to the wells, and the plate was incubated for 1 h at 37 °C. A serial 3 × dilution of the agonist compound in water was made, and 10 µl of the serial dilutions was applied to the cells in duplicate. The starting concentration of agonist in the assay was 10 µM. After 1–2 h of incubation at room temperature, the fluorescence was measured with excitation at 485/20 nm and emission at 528/20 nm with a Synergy HT Biotek reader.
The next day, when the cells were nearly confluent, an equal amount of 2 × Fluo-4 Direct™ calcium reagent loading solution, containing 5 mM probenecid, was added to the wells, and the plate was incubated for 1 h at 37 °C. A serial 3 × dilution of the agonist compound in water was made, and 10 µl of the serial dilutions was applied to the cells in duplicate. The starting concentration of agonist in the assay was 10 µM. After 1–2 h of incubation at room temperature, the fluorescence was measured with excitation at 485/20 nm and emission at 528/20 nm with a Synergy HT Biotek reader.
9
Fluo-4 Direct Calcium Assay of P2RX7
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Changes in intracellular calcium response were measured using Fluo-4 Direct™ Calcium Assay (ThermoFisher Scientific, Paisley, UK) in accordance with the manufacturer's instructions. Briefly, E0771 cells were seeded in a 96-well plate at a density of 15x10 3 cells per well in complete media and left overnight to adhere prior to the assay. Cells were washed with PBS and were incubated with Fluo-4 Direct™ Calcium reagent for 1h at 37°C, with or without 10µM A740003 P2RX7 antagonist (Bio-Techne Ltd, Abingdon, UK). The cells were then stimulated with 100µM 2'(3')-O-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP; Merck Life Sciences, Gillingham, UK) to activate the P2RX7, and the fluorescence measured using FlexStation-3 Multimode Microplate reader (Molecular Devices, Wokingham, UK) with excitation at 494nm and emission at 506nm. Subsequently, the cells were treated with 500nM ionomycin to obtain the maximal fluorescence for normalisation of the data. The data was acquired for 300 seconds, including an initial 20 second baseline reading
10
Calcium Signaling Dynamics Measurement
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Changes in intracellular calcium concentrations were measured using Fluo-4 Direct™ Calcium Assay (ThermoFisher Scientific, UK) in accordance with the manufacturer's instructions. In brief, 15,000 cells were loaded with Fluo-4 Direct™ calcium reagent, left to incubate for 1 hour at 37°C before being stimulated with 100 µM BzATP (Sigma, UK) and then 0.8 µM ionomycin (Sigma, UK). Excitation ratio and emission wavelength were 490 nm and 525 nm respectively.
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