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Amplite® Fluorimetric Calcium Quantitation Kit *Red Fluorescence*

Calcium dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Calcium Quantitation Kit.
Calcium dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Calcium Quantitation Kit.
Calcium dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Calcium Quantitation Kit.
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Calcium is essential for all living organisms, particularly in cell physiology, where movement of the calcium ion Ca2+ into and out of the cytoplasm functions as a signal for many cellular processes. Calcium is the fifth most abundant element by mass in the human body, where it is a common cellular ionic messenger with many functions, and serves also as a structural element in bone. Calcium plays an important role in mediating the constriction and relaxation of blood vessels, nerve impulse transmission, muscle contraction, and hormone secretion. The serum level of calcium is closely regulated within a fairly limited range (9 to 10.5 mg/dL) in the human body. Both hypocalcaemia and hypercal caemia are serious medical disorders. Causes of low calcium levels include chronic kidney failure, vitamin D deficiency, and low blood magnesium levels that can occur in severe alcoholism. Amplite® Calcium Detection Kit using our proprietary red fluorescence probe provides a simple method for detecting calcium in physiology solutions. The fluorescent signal can be easily read by fluorescence microplate reader. The Amplite® Calcium Detection Kit can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation with no separation steps required. The assay can be completed within 30 minutes. With the Amplite® Calcium Detection Kit, we have detected as little as 0.03 mM calcium.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare assay reaction mixture (50 µL)
  2. Add calcium standards or test samples (50 µL)
  3. Incubate at room temperature for 5 - 30 minutes
  4. Monitor the fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components to room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Rhod Red™ stock solution (200X):
Add 50 µL of sterile H2O into the vial of Rhod Red™ Indicator (Component A).


Calcium standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/36360

Prepare a calcium standard by diluting the appropriate amount of the 300 mM Calcium Standard (Component C) into H2O to produce a Calcium concentration ranging from 0 to 3 mM.


Add 25 μL of Rhod Red™ stock solution (200X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.025 mL. Keep from light.


Table 1. Layout of calcium standards and test samples in a solid black 96-well microplate. CS= Calcium Standards (CS7 - CS1, 0.003 to 3 mM), BL=Blank Control, TS=Test Samples. 


Table 2. Reagent composition for each well.

CS1 - CS750 µLSerial Dilutions (0.003 to 3 mM)
BL50 µLH2O
TS50 µLtest sample
  1. Prepare calcium standards (CS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of working solution to each well of calcium standard, blank control, and test samples to make the total calcium assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction for 5 to 30 minutes at room temperature, protected from light.

  4. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm.



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