Mitochondrial reactive oxygen species (ROS) production rate detection kit (fluorescence method)

Cat. No.: M1492773

GRADE & PURITY BioReagent

Overview

  Reactive oxygen species (ROS) are natural by-products of normal oxygen metabolism, including superoxide radicals, hydrogen peroxide, and their downstream products such as peroxides and hydroxides. Studies show that over 95% of ROS in organisms originate from mitochondria. An imbalance leading to oxidative stress is associated with cell growth, proliferation, development, differentiation, aging, apoptosis, and many physiological and pathological processes. Under normal conditions, a balance exists between the intracellular antioxidant defense system and oxygen free radicals, maintaining ROS at low physiological levels. Under pathological conditions, this balance is disrupted, leading to excessive intracellular ROS levels. This can damage mitochondrial enzymes, lipids, and nucleic acids, causing oxidative stress. Additionally, ROS can attack mitochondrial DNA, causing oxidative damage that leads to structural and functional changes such as reduced mitochondrial ATP synthesis and disrupted mitochondrial membrane potential.

  Mitochondrial Reactive Oxygen Species (ROS) Production Rate Assay Kit (Fluorometric Method) provides a simple, sensitive, and rapid method for detecting mitochondrial ROS production rate. The principle utilizes the fluorescent probe DCFH-DA for ROS detection. DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) diffuses across the mitochondrial membrane and is hydrolyzed by esterases inside the mitochondria to form non-fluorescent DCFH. DCFH is then oxidized by ROS to generate fluorescent DCF. The rate of increase in DCF fluorescence intensity is proportional to the rate of ROS production.

M1492773	Component	96T	Storage
M1492773A	Extraction Buffer	60 mL×2	2-8℃
M1492773B	ReagentⅠ	50 mL	2-8℃
M1492773C	Reagent Ⅱ	1.5 mL	-20℃. Store in the dark.
M1492773D	Reagent Ⅲ	1EA	2-8℃. Store in the dark.
M1492773E	Reagent Ⅳ	1EA	2-8℃. Store in the dark.
M1492773F	Reagent Ⅴ	1EA	2-8℃. Store in the dark.
M1492773G	Reagent Ⅵ	20 μL	-20℃. Store in the dark.

Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

User-Provided Instruments and Consumables

Adjustable pipettes and tips

Homogenizer, Low-temperature centrifuge, 96-well solid black or solid white microplate

Constant temperature incubator, Multifunctional microplate reader

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C
ReagentⅠ	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C
ReagentⅡ	Ready-to-use	Store at -20°C protected from light.
ReagentⅢ	Prepare before use: Dissolve contents for 96 tests in 6 mL Reagent I. Mix well.	Unused dissolved Reagent III can be stored at 4°C protected from light for 1 month.
ReagentⅣ	Prepare before use: Dissolve contents for 96 tests in 6 mL Reagent I. Mix well.	Unused dissolved Reagent IV can be stored at 4°C protected from light for 1 month.
ReagentⅤ	Prepare before use: Dissolve contents for 96 tests in 6 mL Reagent I. Mix well.	Unused dissolved Reagent V can be stored at 4°C protected from light for 1 month.
ReagentⅥ		Reagent VI is somewhat irritating; personal protection is recommended during use.
Working ReagentⅥ	Prepare before use: Dilute Reagent VI 300-fold with Reagent I according to the required volume.	Diluted Working Reagent VI cannot be reused.

2. Sample Preparation (Tissue/Cell Mitochondria Extraction)

2.1 Weigh approximately 0.1 g of tissue or collect 5 million cells. Add 1 mL of Extraction Buffer and 10 µL of Reagent II. Homogenize on ice using a homogenizer. Centrifuge at 600 g, 4°C for 5 minutes. Collect the supernatant into a new centrifuge tube, discard the pellet.

2.2 Centrifuge the supernatant again at 11,000 g, 4°C for 10 minutes. The pellet contains the extracted mitochondria.

2.3 Discard the supernatant. Resuspend the pellet in 200 µL of Reagent I. Keep on ice for immediate assay.

Notes:

(1) Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month.

(2) Extracted mitochondrial samples must be assayed on the same day and should not be frozen.

(3) For protein concentration determination, Aladdin B774074 Bradford Protein Assay Kit or B406195 Bradford Assay Solution (Ready-to-Use) [for Protein Determination] is recommended.

3. Assay Steps

3.1 Pre-heat the multifunctional microplate reader to 37°C. Set the fluorescence excitation wavelength to 488 nm and emission wavelength to 525 nm.

3.2 Add reagents to a 96-well solid black or solid white microplate as follows:

Reagent	Blank Well (µL)	Test Well (µL)
Sample	0	20
ReagentⅠ	20	0
ReagentⅢ	50	50
ReagentⅣ	50	50
ReagentⅤ	50	50
Working ReagentⅥ	30	30

3.3 Mix well. Incubate at 37°C protected from light for 15 minutes.

3.4 After incubation, measure the fluorescence intensity over 10 minutes using the microplate reader (Ex/Em = 488/525 nm). Maintain the instrument temperature at 37°C. Record the fluorescence change over 10 minutes.

Notes:

(1) Fluorescence intensity changes must be measured at a constant 37°C over 10 minutes.

(2) When mixing with a pipette, pipette gently to avoid generating bubbles.

(3) Use solid black or white 96-well plates to prevent interference between adjacent wells. 

4. Result Calculation 

4.1 Data Processing 

Perform linear regression analysis on the sampled data points (fluorescence intensity vs. time) to calculate the regression coefficient, i.e., the slope (k) of the line. The actual mitochondrial 

ROS production rate equals the slope (k _test ) from the linear regression of the sample's fluorescence intensity vs. time data points minus the slope (k _blank ) from the linear regression of the background fluorescence intensity vs. time data points. k = (RFU _10min - RFU _0min ) / 600 (assuming time in seconds; 10 min = 600 s) 

4.2 Activity Calculation 

Note: We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas. 

(1) Based on sample mass: 

(1) Based on sample mass: ROS Production Rate (RFU/s/g fresh weight) = (k _test - k _blank ) ÷ (V _sample ÷ V _total × W) = 100 × (k _test - k _blank ) 

(2) Based on sample protein concentration: 

ROS Production Rate (RFU/s/mg prot) = (k _test - k _blank ) ÷ (V _sample ÷ V _total × Cpr) = 10 × (k _test - k _blank ) ÷ Cpr 

(3) Based on cell count: 

ROS Production Rate (RFU/s/10⁴ Cells) = (k _tes t - k _blank ) ÷ (500 × V _sample ÷ V _total ) = (k _test - k _blank ) ÷ 50 

Parameter Description: 

V _sample : Sample volume added, 0.02 mL 

V _total : Total resuspension volume of the sample, 0.2 mL

Cpr: Sample protein concentration, mg/mL

W: Sample mass, 0.1 g

500: Cell count, in units of 10⁴

Precautions

1.Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.

2.This product is for scientific research use only. Not intended for clinical diagnosis.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.