Purified mitochondrial respiratory control rate ( RCR ) quantitative detection kit
The main purpose
Purified mitochondrial respiratory control rate (RCR) quantitative detection reagent is a method for determining the dissolved oxygen in the presence of ADP (III state respiration) or not (IV state respiration) in fresh living mitochondria by polarographic system. The difference in the consumption of dissolved oxygen, the respiratory control rate (RCR), is an authoritative and classical technique for evaluating mitochondrial structure and functional integrity as well as oxidative phosphorylation efficiency. The technology has been carefully developed and successfully tested. It can be used for studies such as mitochondrial physiological functions and drug action mechanisms. The product is strictly sterile, ready to use, easy to operate, in vivo detection, and stable performance.
technical background
Mitochondria are the center of cellular respiratory chain and oxidative phosphorylation. Electron transfer and ATP synthesis are coupled by a proton gradient. The mitochondria are structurally intact, functioning normally, and the substrate is sufficient. The proton gradient formed by electron transfer is continuously consumed, the electrons are smoothly transmitted, the oxygen is rapidly consumed, and the oxygen consumption rate is large, which is the III state breathing. ADP depletion, proton gradient can not be consumed, hinder electron transfer, oxygen consumption is reduced, and IV state breathing. The respiratory control ratio (RSR), also known as the respiratory regulation ratio, refers to the ratio of the respiratory rate of the III state (added to ADP) to the respiratory rate of the IV state (ADP depletion). Normal mitochondria have an RCR of 3 to 10: a decrease in RCR means impaired mitochondrial ATP synthesis and a respiratory disorder; an increase in RCR means that cells are active and metabolism is accelerated.
product content
Medium solution (Reagent A) ml
IV state substrate liquid (Reagent B) microliter
III state substrate liquid (Reagent C) microliter
Product manual 1 copy
storage method
Stored in a -20 ° C refrigerator, effective for June
User-supplied
CLARK oxygen electrode meter: used to determine dissolved oxygen concentration
Experimental procedure
Before the start of the experiment, fresh mitochondria were prepared and placed in an ice bath. At the same time, the reagent in the -20 °C refrigerator was melted and the oxygen electrode was preheated to 25 °C. Then do the following.
Add xx ml of media solution (Reagent A) to the reaction glass cell
- Mix thoroughly with micro-magnetic stirring
- Sealing reaction tank
- Start recording oxygen concentration: initial saturated oxygen concentration value is 0.240 micromoles molecular oxygen / ml (25 ° C)
- After 1 minute of continuous recording, add 20 μl of mitochondria to be tested (total 2 mg) (Note: Oxygen concentration may change instantaneously)
- After 1 minute of continuous recording, add xx μl of IV-state substrate solution (Reagent B) to start IV-state respiration (Note: see note 9 )
- Continuous recording for 2 minutes: a slow drop in oxygen concentration
- Continue to inject xx microliters of Phase III substrate solution (Reagent C) ( Note: oxygen concentration begins to decline within 10 seconds ) - Start III state breathing (Note: see note 9 )
- Continue recording until there is a linear down slash
- Calculate the III state respiratory rate and the IV state respiratory rate separately: oxygen concentration reduction value ÷ actual time (minutes)
- Calculate respiratory control rate: III state respiratory rate ÷ IV state respiratory rate
Precautions
- This product is 20 operations
- Wear gloves when handling
- Ensure that the reaction glass tank is clean and sealed to prevent oxygen from flowing in
- Mitochondrial samples must be prepared fresh and are not recommended for cryopreservation; the prepared mitochondrial membrane must be intact and very important; mitochondrial separation kit is recommended - YIJI10006.1
- Avoid air bubbles and air injection when injection is added to the reaction tank
- Avoid ethanol contamination; ethanol increases oxygen concentration; if substrate or inhibitor must be dissolved in ethanol, add 10 μl
- Keep the temperature of the reaction tank constant, the temperature decreases, and the oxygen concentration rises.
- The reaction solution is thoroughly mixed and balanced with ambient oxygen
- Fully freeze-thaw and mix before adding IV medium solution (Reagent B) and III-state substrate solution (Reagent C)
- Add reagents strictly according to the regulations, and do not increase the filling reagent capacity.
- The company provides a series of mitochondrial detection reagent products
Quality Standard
- This product has been certified to be stable.
- This product has been identified and sensitive
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