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Product key features
- Comprehensive: Includes key assay components needed for COX-2 activity and COX-2 inhibitor screening.
- Precise results: Accurately measures COX-2 activity/inhibition in various sample types.
- Convenient: Ready-to-use reagents for streamlined workflows.
- Versatility: Suitable for cell lysates, purified enzymes, or tissue homogenates.
- Quick assay: Obtain results in less than 30 minutes.
Product description
The Amplite® Fluorometric Cyclooxygenase-2 (COX-2) Activity Assay kit provides a one-stop solution for measuring cyclooxygenase-2 (COX-2) activity and screening inhibitors of COX-2. Cyclooxygenase-2 is one of the two isoenzymes of cyclooxygenase, with COX-1 being constitutively expressed in many tissues. Cox-2 is an inducible enzyme that converts arachidonic acid into prostaglandins, which are lipid mediators involved in inflammation, pain, and cancer. COX-2 is a key target in anti-inflammatory and anti-cancer drug research.
This kit uses a fluorescence detection system to measure COX-2 activity. The enzyme catalyzes the oxidation of a arachidonic acid to generate a fluorogenic product, resulting in a fluorescence signal (Ex/Em = 540/590 nm). The signal generated is directly proportional to the enzymatic activity, enabling accurate quantification. This kit provides a simple and reliable solution for detecting COX-2 activity in a wide range of biological samples, including cell lysates, serum, purified enzymes, and tissue homogenates. It is compatible with fluorescence microplate readers and is ideal for studying enzyme kinetics or disease-related pathways involving COX-2 and for screening COX-2 inhibitors.
Example protocol
AT A GLANCE
Protocol summary
- prepare test samples along with COX-2 positive control, resorufin standard and COX-2 inhibitor
- Add COX-2 working solution (50 μL) in the test sample, positive control and inhibitor well.
- Incubate at 10–30 minutes at RT.
- Monitor fluorescence intensity at Ex/Em = 540 nm/ 590 nm (Cutoff = 570 nm).
Important notes
Store Component C (COX-2 Positive Control) at <-65 oC when received the kit.
Thaw all the kit components (except COX-2 Positive Control thaw at 4 oC) at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTIONS
COX-2 Positive Control:
Add 4 µL COX-2 Positive Control (Components C) to 100 µL of COX-2 Assay Buffer (Component B) to make COX-2 positive control solution. Mix well by pipetting up and down. Do not store diluted COX-2 solution.
Arachidonic Acid Stock Solution:
Mix 1:1 ratio of Arachidonic Acid (Component D) and Substrate Diluent (Component F) (For example: 10 µL Component D mix with 10 µL Component F). This substrate solution should be prepared fresh before the experiment.
COX-2 Probe Stock Solution:
Add 60 µL DMSO to the vial of Component A to make 100X COX-2 probe stock solution.
The COX-2 probe stock solution should be aliquoted and stored at -20°C after preparation. Avoid repeated freeze-thaw cycles.
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
Prepare COX-2 Enzyme Solution:
Add 4µL COX-2 Positive Control (Components C) to 100 µL of COX-2 Assay Buffer (Component B) to make COX-2 Enzyme Solution. Mix well by pipetting up and down.do not store diluted COX-2 solution.
COX-2 Inhibitor Standard Curve:
Add 5 μL of Celecoxib (Component H) to 495 µL of COX-2 Assay Buffer (Component B) to make 100 µM of Inhibitor standard solution (Std7). Take 250 µL (Std7) and perform 1:2 serial dilutions in COX-2 Assay Buffer (Component B) to get serially diluted Inhibitor Standards (Std6-Std1).
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
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/15253
PREPARATION OF WORKING SOLUTION
Add the following components to 5 mL COX-2 Assay Buffer (Component B) in this sequence:
Add 50 µL of COX-2 Probe Stock Solution (Component A), mix well, and then add 20 µL of Arachidonic Acid Stock Solution, mix well, and lastly add 10 µL Cofactor (Component E).
Note:
This COX-2 working solution should be prepared freshly before the experiment, and kept from light.
5mL COX-2 Working Solution is for 100 tests; please prepare the amount of COX-2 Working Solution as needed proportionally.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of Resorufin standards and test samples in a solid black 96-well microplate. STD = Resorufin Standards (STD1-STD7, 0.15 to 10 µM), BL= Blank Control, TS = Test Samples.
| BL | BL | TS | TS |
| STD 1 | STD 1 | COX-2 Positive Control | COX-2 Positive Control |
STD 2 | STD 2 | ... | ... |
STD 3 | STD 3 | ||
STD 4
| STD 4
| ||
STD 5 | STD 5 | ||
STD 6 | STD 6 | ||
STD 7 | STD 7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
| STD1-STD7 | 50 µL | Serial Dilutions (0.15 to 10 µM) |
| BL | 50 µL | COX-2 Assay Buffer (Component B) |
| TS | 50 µL | Test Sample |
| COX-2 Positive Control | 50 µL | COX-2 Positive Control solution |
- Prepare Resorufin standards (STD1-7), blank controls (BL), COX-2 Positive Control 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 COX-2 Working Solution to each well of Test sample, blank control, and COX-2 Positive Control. For a 384-well plate, add 25 µL of COX-2 Working Solution into each well instead.
- Immediately after addition of the COX-2 Working Solution, measure the fluorescence (RFU) using the preset plate reader settings (Ex = 540 nm/Em = 590 nm, Cutoff=570nm) in kinetic mode reading every 30 seconds for a total of 20 minutes at room temperature. The Standard Curve can be read together with the sample or at the end of the incubation time.
Table 3. Layout of Inhibitor standards in a solid black 96-well microplate. STD=Inhibitor (STD1-STD7, 0.15 to 10 µM), BL=Blank Control, TS=Test Samples.
| BL | BL | ... | ... |
| STD 1 | STD 1 | ||
| STD 2 | STD 2 | ||
| STD 3 | STD 3 | ||
| STD 4 | STD 4 | ||
| STD 5 | STD 5 | ||
| STD 6 | STD 6 | ||
| STD 7 | STD 7 |
- Prepare 40 µL COX-2 Enzyme Working solution to (STD1-7), blank controls (BL) according to the layout provided in Table 3. For a 384-well plate, add 20 µL of reagent per well instead of 40 µL.
- Add 10 µL inhibitor solutions to each well, and incubate 5-10 min at room temperature. For a 384-well plate, add 5 µL of reagent per well.
- Add 50 µL of COX-2 Working Solution to each well. For a 384-well plate, add 25 µL of COX-2 Working Solution into each well instead.
- Incubate 20 minutes at room temperature, and read fluorescence intensity at (Ex = 540 nm/Em = 590 nm, Cutoff = 570nm)
References
Authors: Kahk, Nesma M and Mohamed, Fatma E A and Abdelhakeem, Marwa M and Bakr, Rania B
Journal: European journal of medicinal chemistry (2025): 117340
Authors: Afzal, Sana and Iqbal, Mohammad Saeed and Khan, Abdul Haleem
Journal: Current medicinal chemistry (2025)
Authors: Luo, Peng and Chen, Taotao and Huang, Shaoling and Peng, Feng and Huang, Yunhou and Pan, Weigao
Journal: Bioorganic & medicinal chemistry (2025): 118022
Authors: Wang, Yanlong and Peng, Yulin and Hao, Wenjun and He, Chengjian and Gao, Xiang and Liang, Peng and Zhao, Haolin and Wang, Ying and Wang, Liang and Yu, Zhenlong and Liu, Zhiyu
Journal: iScience (2025): 111505
Authors: Kim, Ji-Eun and Wang, Su Hyeon and Lee, Duk-Shin and Kim, Tae-Hyun and Kang, Tae-Cheon
Journal: Brain research (2025): 149345
Product protocol