Amplite® Fluorimetric Calcium Quantitation Kit Red Fluorescence

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

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.

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Solid black

Components

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

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.

Images

Figure 1. Calcium dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Calcium Quantitation Kit.

Citations

View all 5 citations: Citation Explorer

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

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

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

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

High efficiency single-step biomaterial-based microparticle fabrication via template-directed supramolecular coordination chemistry
Authors: Lai, Kwok Kei and Renneberg, Reinhard and Mak, Wing Cheung
Journal: Green Chemistry (2016): 1715--1723

References

View all 105 references: Citation Explorer

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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
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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
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3,4-Dichloropropionanilide (DCPA) inhibits T-cell activation by altering the intracellular calcium concentration following store depletion
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Journal: Toxicol Sci (2008): 97

Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry
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Journal: Naunyn Schmiedebergs Arch Pharmacol (2008): 423

Platelet free cytosolic calcium concentration during ageing of type 2 diabetic patients
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Journal: Platelets (2007): 473

Analysis of calcium concentration fluctuations in hepatopancreatic R cells of Marsupenaeus japonicus during the molting cycle
Authors: Zilli L, Schiavone R, Storelli C, Vilella S.
Journal: Biol Bull (2007): 161

Effect of extracts from safflower seeds on osteoblast differentiation and intracellular calcium ion concentration in MC3T3-E1 cells
Authors: Jang HO, Park YS, Lee JH, Seo JB, Koo KI, Jeong SC, Jin SD, Lee YH, Eom HS, Yun I.
Journal: Nat Prod Res (2007): 787

Measurement of serum calcium concentration after administration of four gadolinium-based contrast agents to human volunteers
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Journal: AJR Am J Roentgenol (2007): 1539