Microplate Assays Using Ion Indicators
Maintaining highly asymmetric concentrations of inorganic cations and anions in a steady state is a feature of living cells. Regulating these ionic gradients is critical for most cellular functions, and measuring ionic concentrations with both spatial and temporal resolution has become critical in research ranging from drug discovery to studies of neuronal function.
We’ve developed a number of Molecular Probes® ion indicators to track calcium and other ion concentrations with intense fluorescent signals and a range of wavelength options.
Explore our portfolio of easy-to-learn, easy-to-use plate readers
Intracellular calcium assays
Calcium flux assays are widely used for in-cell measurement of agonist-stimulated and antagonist-inhibited signaling through G protein–coupled receptors (GPCRs), a large and active target class relevant in drug discovery. The fluo series of calcium indicators emits minimal fluorescence at resting levels of Ca2+, and each increases its fluorescence intensity up to >100-fold with increasing Ca2+ concentration. Cell-permeant formulations can be loaded in cell culture medium and are compatible with imaging and microplate assays, including HTS.
- Also see imaging assays for calcium flux
- Also see high-throughput assays for calcium flux
Intracellular magnesium assays
Intracellular Mg2+ is important for mediating enzymatic reactions, DNA synthesis, hormonal secretion, and muscular contraction. To facilitate the investigation of magnesium’s role in these and other cellular functions, Molecular Probes® fluorescent indicators are designed to deliver ratiometric or intensity-based signals for reporting Mg2+ concentration using UV or visible excitation.
Intracellular pH measurement
Intracellular pH is generally between ~6.8 and 7.4 in the cytosol and ~4.5 and 6.0 in the cell’s acidic organelles. Under physiological conditions, pH inside a cell varies by only fractions of a pH unit, and such changes may occur quite slowly. We offer a range of Molecular Probes® indicators for tracking intracellular pH in the cytosol or in particular organelles.
Dose-dependent calcium response to muscarinic 1 (M1) receptor agonists. CHO M1 cells were plated in a poly-D-lysine coated 384-well plate and incubated overnight. The following day, cells were assayed for a calcium response to carbachol using the Fluo-4 Direct™ Assay. Cells were stimulated with the agonists carbachol, MCN-A-343, bethanechol, oxotremorine, and pilocarpine. Measurements are given in relative fluorescent units, as the maximum response minus the minimum response, divided by the minimum response. Rank order of agonist potency agreed with published results.
Fluo-4 Direct™ Calcium Assay Kit, Starter pack | Fluo-4 NW Calcium Assay Kit, starter pack with buffer | Fluo-4, AM, cell permeant, Special Packaging | Fluo-3, AM, cell permeant, Special Packaging | Fura-2, AM, cell permeant | |
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Target | Calcium flux | Calcium flux | Calcium flux | Calcium flux | Calcium flux |
Reporter | Fluo-4 | Fluo-4 | Fluo-4 | Fluo-3 | Fura-2 |
Ex/Em (nm) | 488/530 | 488/530 | 488/530 | 506/526 | 350,380/510 (ratiometric excitation) |
Assay | Easiest assay for intracellular calcium in the presence of complete culture media | Simplified assay for intracellular calcium without a wash step | Assay for intracellular calcium | Traditional assay for intracellular calcium | Ratiometric and UV light–excitable measurement for intracellular calcium |
Usage | Addition only assay format does not require cell washing or media removal | Requires media removal but no cell washing | Requires media removal and cell washing or quenching | Requires media removal and cell washing or quenching | Requires cell loading |
Components | Includes reagent, PowerLoad™ reagent, and buffer | Includes reagent, PowerLoad™ reagent, and buffer | Bulk reagent | Bulk reagent | Bulk reagent |
Format | 1 kit, 20 plates | 1 kit, 10 plates | 20 x 50 µg | 10 x 50 µg | 20 x 50 µg |
Protocol outline |
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Cat. No. | F10471 | F36206 | F14201 | F1242 | F1221 |
Mag-Fura-2, AM (cell permeant) | Magnesium Green™, AM (cell permeant) | |
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Target | Mg2+ | Mg2+ |
Reporter | Mag-Fura-2 | Magnesium Green™ |
Ex/Em (nm) | 330/491 to 369/511 | 506/531 nm |
Sample | Medium Mg2+ concentration | Lowest Mg2+ concentration |
Usage | Ratio assay with lower affinity for Mg2+ (Kd ~1.9 mM) than Magnesium Green™ | Intensity assay with higher affinity for Mg2+ (Kd ~1.0 mM) than Mag-Fura-2 (Kd ~1.9 mM) |
Components | Bulk reagent | Bulk reagent |
Format | 20 x 50 µg | 20 x 50 µg |
Protocol outline |
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Cat. No. | M1292 | M3735 |
pHrodo™ Green AM Intracellular pH indicator | pHrodo™ Red AM Intracellular pH indicator | 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) | |
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Target | Intracellular pH | Intracellular pH | Intracellular pH |
Reporter | pHrodo™ Green | pHrodo™ Red | BCECF |
Ex/Em (nm) | 509/533 | 560/585 | 440/490,535 |
Sample | Load cells in media | Load cells in media | Load cells in media |
Usage | Green fluorescence increases from pH 8 to pH 4 | Red fluorescence increases from pH 8 to pH 4 | Ratio of emission intensity is pH-dependent from 6.2 to 9.5 |
Components | Bulk reagent | Bulk reagent | Bulk reagent |
Format | 50 µL of 5 mM (1,000x) | 50 µL of 5 mM (1,000x) | 20 x 50 µg |
Protocol outline |
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Cat. No. | P35373 | P35372 | B1170 |
Fluo-4 Direct™ Calcium Assay Kit, Starter pack | Fluo-4 NW Calcium Assay Kit, starter pack with buffer | Fluo-4, AM, cell permeant, Special Packaging | Fluo-3, AM, cell permeant, Special Packaging | Fura-2, AM, cell permeant | |
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Target | Calcium flux | Calcium flux | Calcium flux | Calcium flux | Calcium flux |
Reporter | Fluo-4 | Fluo-4 | Fluo-4 | Fluo-3 | Fura-2 |
Ex/Em (nm) | 488/530 | 488/530 | 488/530 | 506/526 | 350,380/510 (ratiometric excitation) |
Assay | Easiest assay for intracellular calcium in the presence of complete culture media | Simplified assay for intracellular calcium without a wash step | Assay for intracellular calcium | Traditional assay for intracellular calcium | Ratiometric and UV light–excitable measurement for intracellular calcium |
Usage | Addition only assay format does not require cell washing or media removal | Requires media removal but no cell washing | Requires media removal and cell washing or quenching | Requires media removal and cell washing or quenching | Requires cell loading |
Components | Includes reagent, PowerLoad™ reagent, and buffer | Includes reagent, PowerLoad™ reagent, and buffer | Bulk reagent | Bulk reagent | Bulk reagent |
Format | 1 kit, 20 plates | 1 kit, 10 plates | 20 x 50 µg | 10 x 50 µg | 20 x 50 µg |
Protocol outline |
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Cat. No. | F10471 | F36206 | F14201 | F1242 | F1221 |
Mag-Fura-2, AM (cell permeant) | Magnesium Green™, AM (cell permeant) | |
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Target | Mg2+ | Mg2+ |
Reporter | Mag-Fura-2 | Magnesium Green™ |
Ex/Em (nm) | 330/491 to 369/511 | 506/531 nm |
Sample | Medium Mg2+ concentration | Lowest Mg2+ concentration |
Usage | Ratio assay with lower affinity for Mg2+ (Kd ~1.9 mM) than Magnesium Green™ | Intensity assay with higher affinity for Mg2+ (Kd ~1.0 mM) than Mag-Fura-2 (Kd ~1.9 mM) |
Components | Bulk reagent | Bulk reagent |
Format | 20 x 50 µg | 20 x 50 µg |
Protocol outline |
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Cat. No. | M1292 | M3735 |
pHrodo™ Green AM Intracellular pH indicator | pHrodo™ Red AM Intracellular pH indicator | 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) | |
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Target | Intracellular pH | Intracellular pH | Intracellular pH |
Reporter | pHrodo™ Green | pHrodo™ Red | BCECF |
Ex/Em (nm) | 509/533 | 560/585 | 440/490,535 |
Sample | Load cells in media | Load cells in media | Load cells in media |
Usage | Green fluorescence increases from pH 8 to pH 4 | Red fluorescence increases from pH 8 to pH 4 | Ratio of emission intensity is pH-dependent from 6.2 to 9.5 |
Components | Bulk reagent | Bulk reagent | Bulk reagent |
Format | 50 µL of 5 mM (1,000x) | 50 µL of 5 mM (1,000x) | 20 x 50 µg |
Protocol outline |
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Cat. No. | P35373 | P35372 | B1170 |
Expertly detect fluorescence with Thermo Scientific plate readers
High-sensitivity fluorescence detection for 96-1,536 samples can be quickly performed on Fluoroskan or Fluoroskan FL Microplate Fluorometer or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents for optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.
Resources
Molecular Probes® Handbook
- Fluorescent Indicators for Ca2+, Mg2+, Zn2+, and Other Metal Ions—Chapter 19
- Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3
- Fluorescent Mg2+ Indicators—Section 19.6
- pH Indicators—Chapter 20
More information
- Learn more about Calcium Indicators in Drug Discovery
For Research Use Only. Not for use in diagnostic procedures.