Amplite® Fluorimetric Calcium Quantitation Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
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
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
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
Citations
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: Furukawa, Kyohei and Kono, Masaya and Kataoka, Tetsuro and Hasebe, Yukio and Jia, Huijuan and Kato, Hisanori
Journal: Nutrients (2021): 2144
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: Wang, Xueting and Ding, Jie and Xiang, Zou and Jiang, Peipei and Du, Jing and Han, Xiaodong
Journal: Toxicon (2016): 45--55
Authors: Lai, Kwok Kei and Renneberg, Reinhard and Mak, Wing Cheung
Journal: Green Chemistry (2016): 1715--1723
References
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Application notes
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