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AAT Bioquest

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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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

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.

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. 

BLBLTSTS
CS1CS1......
CS2CS2......
CS3CS3  
CS4CS$  
CS5CS5  
CS6CS6  
CS7CS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
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.

Images


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
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
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
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
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
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
Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry
Authors: Asai M, Takeuchi K, Uchida S, Urushida T, Katoh H, Satoh H, Yamada S, Hayashi H, Watanabe H.
Journal: Naunyn Schmiedebergs Arch Pharmacol (2008): 423
Platelet free cytosolic calcium concentration during ageing of type 2 diabetic patients
Authors: Alex, undefined and ru N, Popov D, Sbarcea A, Amuzescu M.
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
Authors: Brown JJ, Hynes MR, Wible JH, Jr.
Journal: AJR Am J Roentgenol (2007): 1539