Amplite® Fluorimetric Hypochlorite (Hypochlorous Acid) Assay Kit
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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 |
Excitation (nm) | 555 |
Emission (nm) | 576 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Excitation (nm) 555 | Emission (nm) 576 |
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 Hypochlorite working solution (50 µL)
- Add Hypochlorite standards or test samples (50 µL)
- Incubate at room temperature for 10 - 30 min
- Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)
Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Oxirite™ Hypochlorite Sensor stock solution (200X):
Add 50 µL of DMSO (Component D) into the vial of Oxirite™ Hypochlorite Sensor (Component A) to make 200X stock solution. Protect from light.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13846
Add 50 µL of Hypochlorite Standard (Component C) into 450 µL of Assay Buffer (Component B) to get 100 mM Hypochlorite standard solution (H7). Take 100 mM (H7) Hypochlorite standard solution and perform 1:10 serial dilutions in Assay Buffer (Component B) to get 10, 1, 0.1, and 0.01 mM serially diluted Hypochlorite standards (H6 - H3). Then, 0.01 mM Hypochlorite standard (H3) and perform 1:3 in Assay Buffer (Component B) to get 0.003 and 0.001 mM diluted Hypoclorite standards (H2 - H1).
PREPARATION OF WORKING SOLUTION
Add 25 µL of 200X Oxirite™ Hypochlorite Sensor stock solution into 5 mL of Assay Buffer (Component B) and mix well to make Hypochlorite working solution. Note: This Hypochlorite working solution is enough for one 96-well plate. It is not stable, use it promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of Hypochlorite standards and test samples in a 96-well solid black microplate. H= Hypochlorite Standards (H1 - H7, 0.001 to 100 mM), BL=Blank Control, TS=Test Samples.
BL | BL | TS | TS |
H1 | H1 | ... | ... |
H2 | H2 | ... | ... |
H3 | H3 | ||
H4 | H4 | ||
H5 | H5 | ||
H6 | H6 | ||
H7 | H7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
H1 - H7 | 50 µL | Serial Dilutions (0.001 to 100 mM) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | test sample |
- Prepare Hypochlorite standards (H), 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 Hypochlorite working solution to each well of Hypochlorite standard, blank control, and test samples to make the total Hypochlorite assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Hypochlorite working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
Images
Citations
Authors: Kavanaugh, Taylor Elizabeth
Journal: (2017)
References
Authors: Martin DE, De Almeida JF, Henry MA, Khaing ZZ, Schmidt CE, Teixeira FB, Diogenes A.
Journal: J Endod (2014): 51
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Journal: J Toxicol Sci (2014): 71
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Journal: Lett Appl Microbiol (2013): 259
Authors: Zhang J, Yang X.
Journal: Analyst (2013): 434
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Journal: Exp Anim (2013): 237
Authors: Perez-Cruz F, Cortes C, Atala E, Bohle P, Valenzuela F, Olea-Azar C, Speisky H, Aspee A, Lissi E, Lopez-Alarcon C, Bridi R.
Journal: Molecules (2013): 1638
Authors: Kanno A, Kawakami T, Takahashi Y, Onodera S.
Journal: J Toxicol Sci (2012): 389
Authors: Zhang J, Wang X, Yang X.
Journal: Analyst (2012): 2806
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