Calcium 4 assay kit

Manufactured by Molecular Devices

The Calcium 4 Assay kit is a fluorescence-based reagent system designed for the detection and measurement of calcium levels in various sample types. The kit provides a sensitive and reliable method to quantify calcium concentrations without the need for complex sample preparation.

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Lab products found in correlation

Prism 5, by GraphPad (2 mentions) Flexstation ii384, by Molecular Devices (2 mentions) Poly d lysine 384 well plate, by Greiner (2 mentions) Chm 271, by Thermo Fisher Scientific (2 mentions) Rabbit anti human ig fitc, by Merck Group (1 mentions) Probenecid, by Merck Group (1 mentions) Saccharin sodium salt hydrate, by Merck Group (1 mentions) Phenylthiourea, by Merck Group (1 mentions) C4 hsl, by Cayman Chemical (1 mentions)

Spelling variants of this product

FLIPR Calcium 4 Assay Kit (26 citations) Fluo-4 NW Calcium Assay Kit (2 citations)

4 protocols using calcium 4 assay kit

Antibody Characterization for P2X Receptors

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Cell surface binding: HEK cells expressing human or rat P2X3, or human P2X2, and control cells were incubated with antibody supernatants for 1 h on ice, washed and incubated with rabbit anti-human Ig FITC (Sigma) for 1 h on ice, washed and analyzed by flow cytometry.
Inhibition of Ca⁺⁺ signaling: Antibodies were tested for ability to inhibit α,β-meATP-induced calcium flux using a cell-based Ca²⁺ flux assay [41 (link)]. Briefly, canine Cf2Th cells were transfected with the appropriate expression vector in poly-lysine coated, black 384-well plates with clear bottoms (Costar). Cells were incubated for 22 h at 37°C then washed twice and loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit, Molecular Devices), incubated for 1.5 h, then moved to a Flexstation II-384 (Molecular Devices) set at 32°C. After a 10-min temperature equilibration, the specific P2X3 agonist α,β-meATP (Sigma) was injected (at t = 20 s) and fluorescence was measured for 60 s (reading every 3 s). Data sets were analyzed using Prism 5.0 software (GraphPad Software, Inc).

Mettler Izquierdo S., Varela S., Park M., Collarini E.J., Lu D., Pramanick S., Rucker J., Lopalco L., Etches R, & Harriman W. (2016). High-efficiency antibody discovery achieved with multiplexed microscopy. Microscopy, 65(4), 341-352.

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Calcium Flux Assay with THP-1 Cells

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Example 12

This example illustrates a calcium assay with human THP-1 cells. THP-1 cells (0.3×10⁵cells in 25 μl of the assay buffer HBSS plus 20 mM HEPES) were pre-incubated with serial diluted doses of mAbs for 20 min at room temperature. 25 μl of the pre-incubated cells were added to 25 μl of Calcium 4 Assay kit (Molecular Device # R8142) including 5 mM probenecid (Sigma # P8761-25G) in a Poly-D-Lysine 384-well plate (Greiner #781946). After 1 h incubation at 37° C. 5% CO₂followed by 15 minutes at room temperature and a brief centrifugation for the plate, a 12.5 μl of 5× concentrated ligand was added to each well of the plate as the challenge agonist during detection on the FlexStation3. The final concentration of ligands used in the assay was 2.5 nM of MCP-1 (PROSPEC # CHM-271), 22 nM of MCP-3 (PeproTech #300-17) and 0.43 nM of MCP-5 (PeproTech #250-04). IC₅₀value of each mAb indicated in Table 5 was calculated by Prism5.

The results in Table 5 show the IC₅₀of the each mAb against the corresponding ligand. Each IC₅₀value represents Mean±S.D. (n=2) and one of two independent experiments which had similar results.

TABLE 5

IC₅₀[pM] of mAbs in calcium flux assay using human THP-1 cells

MLN1202

D11B21G11D1H1/45H8L1(Takeda)

MCP-1180920750370250

MCP-3190290250190110

MCP-5280890560270180

US10696746B2. Antigen binding proteins that bind CCR2 (2020-06-30). Sorrento Therapeutics, Inc. [US]. Inventors: Dingqiu Huang [US], Barbara A. Swanson [US], John Dixon Gray [US], Heyue Zhou [US], Guodi Lu [US].

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Calcium Flux Assay with THP-1 Cells

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Example 12

This example illustrates a calcium assay with human THP-1 cells. THP-1 cells (0.3×10⁵cells in 25 μl of the assay buffer HBSS plus 20 mM HEPES) were pre-incubated with serial diluted doses of mAbs for 20 min at room temperature. 25 μl of the pre-incubated cells were added to 25 μl of Calcium 4 Assay kit (Molecular Device #R8142) including 5 mM probenecid (Sigma #P8761-25G) in a Poly-D-Lysine 384-well plate (Greiner #781946). After 1 h incubation at 37° C. 5% CO₂followed by 15 minutes at room temperature and a brief centrifugation for the plate, a 12.5 μl of 5× concentrated ligand was added to each well of the plate as the challenge agonist during detection on the FlexStation3. The final concentration of ligands used in the assay was 2.5 nM of MCP-1 (PROSPEC # CHM-271), 22 nM of MCP-3 (PeproTech #300-17) and 0.43 nM of MCP-5 (PeproTech #250-04). IC₅₀value of each mAb indicated in Table 5 was calculated by Prism5.

TABLE 5

IC₅₀[pM] of mAbs in calcium flux assay

using human THP-1 cells

MLN1202

D11B21G11D1H1/45H8L1(Takeda)

MCP-1180920750370250

MCP-3190290250190110

MCP-5280890560270180

US09951138B2. Antigen binding proteins that bind CCR2 (2018-04-24). Sorrento Therapeutics, Inc. [US]. Inventors: Dingqiu Huang [US], Barbara A. Swanson [US], John Dixon Gray [US], Heyue Zhou [US], Guodi Lu [US].

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TAS2R Receptor Function Assay

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Wild-type TAS2R receptors were tested for function using a cell-based Ca²⁺ flux assay described previously [12 (link)], using the following compounds: probenecid, phenylthiourea (PTC), saccharin sodium salt hydrate (all from Sigma, St. Louis, MO), denatonium benzoate and aloin (Alfa Aesar, Ward Hill, MA), 6-n-propylthiouracil (PROP) (Selleck Chemicals) and C4-HSL (N-butyryl-L-homoserine; Cayman Chemical). Briefly, cells were transfected with the appropriate expression vector and a plasmid expressing a Gα16 chimera (Gα16-gust44), containing the last 44 amino acids of rat gustducin) in poly-lysine coated, black 384-well plates with clear bottoms (Costar). Cells were incubated for 22 hours at 37 °C then washed twice and loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit, Molecular Devices), incubated for 1.5 hours, then moved to a Flexstation II-384 (Molecular Devices) set at 32 °C. probenecid, a commonly used additive designed to improve dye-loading of cells, was not included for most incubations due to our previous demonstration of probenecid as a TAS2R16 inhibitor [12 (link)]. After a 10-minute temperature equilibration, ligand was injected (at t = 20 seconds) and fluorescence was measured for 60 seconds (reading every 3 seconds). Data sets were analyzed using Prism 5.0 software (GraphPad Software, Inc).

Sandau M.M., Goodman J.R., Thomas A., Rucker J.B, & Rawson N.E. (2015). A functional comparison of the domestic cat bitter receptors Tas2r38 and Tas2r43 with their human orthologs. BMC Neuroscience, 16, 33.

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