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 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
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
  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. F10471 F36206 F14201 F1242 F1221
  Mag-Fura-2, AM (cell permeant) Magnesium Green™, AM (cell permeant)
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
  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. 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)
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
  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. P35373 P35372 B1170
Intracellular calcium assays
  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
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
  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. F10471 F36206 F14201 F1242 F1221
Intracellular magnesium assays
  Mag-Fura-2, AM (cell permeant) Magnesium Green™, AM (cell permeant)
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
  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. M1292 M3735
Intracellular pH indicators
  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)
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
  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. P35373 P35372 B1170
Thermo Scientific plate readers

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.

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Resources

Molecular Probes® Handbook

More information

For Research Use Only. Not for use in diagnostic procedures.