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Amplite® Fluorimetric Calcium Quantitation Kit *Red Fluorescence*
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.
Example protocol
AT A GLANCE
Protocol summary
- Prepare assay reaction mixture (50 µL)
- Add calcium standards or test samples (50 µL)
- Incubate at room temperature for 5 - 30 minutes
- Monitor the fluorescence intensity at Ex/Em = 540/590 nm
Important notes
Thaw all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
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).
PREPARATION OF STANDARD SOLUTION
Calcium standard
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/36360
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.
PREPARATION OF WORKING SOLUTION
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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
| BL | BL | TS | TS |
| CS1 | CS1 | ... | ... |
| CS2 | CS2 | ... | ... |
| CS3 | CS3 | ||
| CS4 | CS$ | ||
| CS5 | CS5 | ||
| CS6 | CS6 | ||
| CS7 | CS7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| CS1 - CS7 | 50 µL | Serial Dilutions (0.003 to 3 mM) |
| BL | 50 µL | H2O |
| TS | 50 µL | test sample |
- 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.
- 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.
- Incubate the reaction for 5 to 30 minutes at room temperature, protected from light.
- Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm.
Citations
View all 6 citations: Citation Explorer
Ca2+ transients on the T cell surface trigger rapid integrin activation in a timescale of seconds
Authors: Li, Yue and Wang, ShiHui and Zhang, YouHua and Liu, ZhaoYuan and Zheng, YunZhe and Zhang, Kun and Chen, ShiYang and Lv, XiaoYing and Huang, MengWen and Pan, XingChao and others,
Journal: Nature Communications (2024): 1--16
Authors: Li, Yue and Wang, ShiHui and Zhang, YouHua and Liu, ZhaoYuan and Zheng, YunZhe and Zhang, Kun and Chen, ShiYang and Lv, XiaoYing and Huang, MengWen and Pan, XingChao and others,
Journal: Nature Communications (2024): 1--16
The CLOCK protein regulates insulin secretion related with L-type calcium channels in rat pancreatic beta cells
Authors: Tian, Linlin and Li, Xiaodong and Ding, Yi and Li, Minli and Tang, Yunzhao and Li, Daiqing
Journal: Biochemical and biophysical research communications (2022): 116--122
Authors: Tian, Linlin and Li, Xiaodong and Ding, Yi and Li, Minli and Tang, Yunzhao and Li, Daiqing
Journal: Biochemical and biophysical research communications (2022): 116--122
Effects of Eggshell Membrane on Keratinocyte Differentiation and Skin Aging In Vitro and In Vivo
Authors: Furukawa, Kyohei and Kono, Masaya and Kataoka, Tetsuro and Hasebe, Yukio and Jia, Huijuan and Kato, Hisanori
Journal: Nutrients (2021): 2144
Authors: Furukawa, Kyohei and Kono, Masaya and Kataoka, Tetsuro and Hasebe, Yukio and Jia, Huijuan and Kato, Hisanori
Journal: Nutrients (2021): 2144
Realgar transforming solution as a novel arsenic agent with a lower risk of cardiotoxicity
Authors: Hai, Yang and Song, Peng and Wang, Xin and Zhao, Longhe and Xie, Qinjian and Li, Jianyin and Li, Yang and Li, Hongyu
Journal: Journal of pharmacological sciences (2019): 162--170
Authors: Hai, Yang and Song, Peng and Wang, Xin and Zhao, Longhe and Xie, Qinjian and Li, Jianyin and Li, Yang and Li, Hongyu
Journal: Journal of pharmacological sciences (2019): 162--170
Microcystin-LR causes sexual hormone disturbance in male rat by targeting gonadotropin-releasing hormone neurons
Authors: Wang, Xueting and Ding, Jie and Xiang, Zou and Jiang, Peipei and Du, Jing and Han, Xiaodong
Journal: Toxicon (2016): 45--55
Authors: Wang, Xueting and Ding, Jie and Xiang, Zou and Jiang, Peipei and Du, Jing and Han, Xiaodong
Journal: Toxicon (2016): 45--55
References
View all 105 references: Citation Explorer
Calcium, magnesium, iron, zinc and copper concentration in the hair of tobacco smokers
Authors: Unkiewicz-Winiarczyk A, Bagniuk A, Gromysz-Kalkowska K, Szubartowska E.
Journal: Biol Trace Elem Res (2009): 152
Authors: Unkiewicz-Winiarczyk A, Bagniuk A, Gromysz-Kalkowska K, Szubartowska E.
Journal: Biol Trace Elem Res (2009): 152
Genetic and nongenetic variation in concentration of selenium, calcium, potassium, zinc, magnesium, and phosphorus in milk of Dutch Holstein-Friesian cows
Authors: van Hulzen KJ, Sprong RC, van der Meer R, van Arendonk JA.
Journal: J Dairy Sci (2009): 5754
Authors: van Hulzen KJ, Sprong RC, van der Meer R, van Arendonk JA.
Journal: J Dairy Sci (2009): 5754
Comparison of calcium, magnesium, sodium, potassium, zinc, and creatinine concentration in 24-h and spot urine samples in women
Authors: Ilich JZ, Blanusa M, Orlic ZC, Orct T, Kostial K.
Journal: Clin Chem Lab Med (2009): 216
Authors: Ilich JZ, Blanusa M, Orlic ZC, Orct T, Kostial K.
Journal: Clin Chem Lab Med (2009): 216
Atomic absorption spectrometry and scanning electron microscopy evaluation of concentration of calcium ions and smear layer removal with root canal chelators
Authors: Spano JC, Silva RG, Guedes DF, Sousa-Neto MD, Estrela C, Pecora JD.
Journal: J Endod (2009): 727
Authors: Spano JC, Silva RG, Guedes DF, Sousa-Neto MD, Estrela C, Pecora JD.
Journal: J Endod (2009): 727
3,4-Dichloropropionanilide (DCPA) inhibits T-cell activation by altering the intracellular calcium concentration following store depletion
Authors: Lewis TL, Brundage KM, Brundage RA, Barnett JB.
Journal: Toxicol Sci (2008): 97
Authors: Lewis TL, Brundage KM, Brundage RA, Barnett JB.
Journal: Toxicol Sci (2008): 97
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