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.

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.

Fluo-4 Direct™ assay
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.

Selection guides
 Fluo-4 Direct Calcium Assay Kit, Starter packFluo-4 NW Calcium Assay Kit, starter pack with bufferFluo-4, AM, cell permeant, Special PackagingFluo-3, AM, cell permeant, Special PackagingFura-2, AM, cell permeant
TargetCalcium fluxCalcium fluxCalcium fluxCalcium fluxCalcium flux
ReporterFluo-4Fluo-4Fluo-4Fluo-3Fura-2
Ex/Em (nm)488/530488/530488/530506/526350,380/510 (ratiometric excitation)
AssayEasiest assay for intracellular calcium in the presence of complete culture mediaSimplified assay for intracellular calcium without a wash stepAssay for intracellular calciumTraditional assay for intracellular calciumRatiometric and UV light–excitable measurement for intracellular calcium
UsageAddition only assay format does not require cell washing or media removalRequires media removal but no cell washingRequires media removal and cell washing or quenchingRequires media removal and cell washing or quenchingRequires cell loading
ComponentsIncludes reagent, PowerLoad reagent, and bufferIncludes reagent, PowerLoad reagent, and bufferBulk reagentBulk reagentBulk reagent
Format1 kit, 20 plates1 kit, 10 plates20 x 50 µg10 x 50 µg20 x 50 µg
Protocol outline
  1. Plate cells in microplate wells.
  2. Prepare Fluo-4 Direct loading solution.
  3. Add loading solution to wells.
  4. Incubate for 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare dye loading solution.
  2. Remove media from cells.
  3. Add dye loading solution.
  4. Incubate for 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare dye working solution.
  2. Add to cells, incubate 30 min at 37°C.
  3. Wash cells to remove dye.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare dye working solution.
  2. Add to cells, incubate 30 min at 37°C.
  3. Wash cells to remove dye.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare reagent stock solution.
  2. Add to cells, incubate 60 min at 37°C.
  3. Wash cells, add media, incubate 30 min at 37°C.
  4. After loading is complete, perform treatment.
  5. Measure fluorescence at ~510 nm.
  6. Determine fluorescence ratio.
Cat. No.F10471F36206F14201F1242F1221
 Mag-Fura-2, AM (cell permeant)Magnesium Green, AM (cell permeant)
TargetMg2+Mg2+
ReporterMag-Fura-2Magnesium Green
Ex/Em (nm)330/491 to 369/511506/531 nm
SampleMedium Mg2+ concentrationLowest Mg2+ concentration
UsageRatio assay with lower affinity for Mg2+ (Kd ~1.9 mM) than Magnesium GreenIntensity assay with higher affinity for Mg2+ (Kd ~1.0 mM) than Mag-Fura-2 (Kd ~1.9 mM)
ComponentsBulk reagentBulk reagent
Format20 x 50 µg20 x 50 µg
Protocol outline
  1. Prepare dye working solution.
  2. Add to cells.
  3. Incubate for 30 min at 37°C.
  4. Wash cells 3X.
  5. Incubate 30 min at 37°C.
  6. Treat cells.
  7. Measure fluorescence.
  1. Prepare dye working solution.
  2. Add to cells.
  3. Incubate for 30 min at 37°C.
  4. Wash cells 3X.
  5. Incubate 30 min at 37°C.
  6. Treat cells.
  7. Measure fluorescence.
Cat. No.M1292M3735
 pHrodo Green AM Intracellular pH indicatorpHrodo Red AM Intracellular pH indicator2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM)
TargetIntracellular pHIntracellular pHIntracellular pH
ReporterpHrodo GreenpHrodo RedBCECF
Ex/Em (nm)509/533560/585440/490,535
SampleLoad cells in mediaLoad cells in mediaLoad cells in media
UsageGreen fluorescence increases from pH 8 to pH 4Red fluorescence increases from pH 8 to pH 4Ratio of emission intensity is pH-dependent from 6.2 to 9.5
ComponentsBulk reagentBulk reagentBulk reagent
Format50 µL of 5 mM (1,000x)50 µL of 5 mM (1,000x)20 x 50 µg
Protocol outline
  1. Prepare pHrodo dye solution.
  2. Remove media, wash cells.
  3. Add pHrodo dye solution.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Analyze cells.
  1. Prepare pHrodo dye solution.
  2. Remove media, wash cells.
  3. Add pHrodo dye solution.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Analyze cells.
  1. Prepare dye loading solution.
  2. Remove media, wash cells.
  3. Add dye loading solution.
  4. Incubate 30 min at 37°C.
  5. Remove loading solution, wash cells.
  6. Replace media.
  7. Treat cells.
  8. Analyze cells.
Cat. No.P35373P35372B1170
Intracellular calcium assays
 Fluo-4 Direct Calcium Assay Kit, Starter packFluo-4 NW Calcium Assay Kit, starter pack with bufferFluo-4, AM, cell permeant, Special PackagingFluo-3, AM, cell permeant, Special PackagingFura-2, AM, cell permeant
TargetCalcium fluxCalcium fluxCalcium fluxCalcium fluxCalcium flux
ReporterFluo-4Fluo-4Fluo-4Fluo-3Fura-2
Ex/Em (nm)488/530488/530488/530506/526350,380/510 (ratiometric excitation)
AssayEasiest assay for intracellular calcium in the presence of complete culture mediaSimplified assay for intracellular calcium without a wash stepAssay for intracellular calciumTraditional assay for intracellular calciumRatiometric and UV light–excitable measurement for intracellular calcium
UsageAddition only assay format does not require cell washing or media removalRequires media removal but no cell washingRequires media removal and cell washing or quenchingRequires media removal and cell washing or quenchingRequires cell loading
ComponentsIncludes reagent, PowerLoad reagent, and bufferIncludes reagent, PowerLoad reagent, and bufferBulk reagentBulk reagentBulk reagent
Format1 kit, 20 plates1 kit, 10 plates20 x 50 µg10 x 50 µg20 x 50 µg
Protocol outline
  1. Plate cells in microplate wells.
  2. Prepare Fluo-4 Direct loading solution.
  3. Add loading solution to wells.
  4. Incubate for 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare dye loading solution.
  2. Remove media from cells.
  3. Add dye loading solution.
  4. Incubate for 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare dye working solution.
  2. Add to cells, incubate 30 min at 37°C.
  3. Wash cells to remove dye.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare dye working solution.
  2. Add to cells, incubate 30 min at 37°C.
  3. Wash cells to remove dye.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Measure fluorescence.
  1. Prepare reagent stock solution.
  2. Add to cells, incubate 60 min at 37°C.
  3. Wash cells, add media, incubate 30 min at 37°C.
  4. After loading is complete, perform treatment.
  5. Measure fluorescence at ~510 nm.
  6. Determine fluorescence ratio.
Cat. No.F10471F36206F14201F1242F1221
Intracellular magnesium assays
 Mag-Fura-2, AM (cell permeant)Magnesium Green, AM (cell permeant)
TargetMg2+Mg2+
ReporterMag-Fura-2Magnesium Green
Ex/Em (nm)330/491 to 369/511506/531 nm
SampleMedium Mg2+ concentrationLowest Mg2+ concentration
UsageRatio assay with lower affinity for Mg2+ (Kd ~1.9 mM) than Magnesium GreenIntensity assay with higher affinity for Mg2+ (Kd ~1.0 mM) than Mag-Fura-2 (Kd ~1.9 mM)
ComponentsBulk reagentBulk reagent
Format20 x 50 µg20 x 50 µg
Protocol outline
  1. Prepare dye working solution.
  2. Add to cells.
  3. Incubate for 30 min at 37°C.
  4. Wash cells 3X.
  5. Incubate 30 min at 37°C.
  6. Treat cells.
  7. Measure fluorescence.
  1. Prepare dye working solution.
  2. Add to cells.
  3. Incubate for 30 min at 37°C.
  4. Wash cells 3X.
  5. Incubate 30 min at 37°C.
  6. Treat cells.
  7. Measure fluorescence.
Cat. No.M1292M3735
Intracellular pH indicators
 pHrodo Green AM Intracellular pH indicatorpHrodo Red AM Intracellular pH indicator2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM)
TargetIntracellular pHIntracellular pHIntracellular pH
ReporterpHrodo GreenpHrodo RedBCECF
Ex/Em (nm)509/533560/585440/490,535
SampleLoad cells in mediaLoad cells in mediaLoad cells in media
UsageGreen fluorescence increases from pH 8 to pH 4Red fluorescence increases from pH 8 to pH 4Ratio of emission intensity is pH-dependent from 6.2 to 9.5
ComponentsBulk reagentBulk reagentBulk reagent
Format50 µL of 5 mM (1,000x)50 µL of 5 mM (1,000x)20 x 50 µg
Protocol outline
  1. Prepare pHrodo dye solution.
  2. Remove media, wash cells.
  3. Add pHrodo dye solution.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Analyze cells.
  1. Prepare pHrodo dye solution.
  2. Remove media, wash cells.
  3. Add pHrodo dye solution.
  4. Incubate 30 min at 37°C.
  5. Treat cells.
  6. Analyze cells.
  1. Prepare dye loading solution.
  2. Remove media, wash cells.
  3. Add dye loading solution.
  4. Incubate 30 min at 37°C.
  5. Remove loading solution, wash cells.
  6. Replace media.
  7. Treat cells.
  8. Analyze cells.
Cat. No.P35373P35372B1170

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Molecular Probes Handbook

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