Tissue ADP/ATP Ratio Bioluminescence Detection Kit Instructions

Tissue ADP/ATP Ratio Bioluminescence Detection Kit Product Manual (Chinese version)

The main purpose

Tissue ADP/ATP ratio bioluminescence detection reagent is a kind of luminescence luciferase reaction system, fluorescein reacts with ATP under the catalysis of enzyme to produce bio-light energy, and its relative luminescence unit is determined by luminescence meter. Changes to quantify the authoritative and classical technical approach to ADP/ATP ratios in tissue samples (including lysis or suspension) obtained before and after pyruvate kinase treatment. The technology has been carefully developed and successfully tested. It is suitable for ADP/ATP ratio detection of various tissue (animal, human) samples. The product is strictly sterile, ready to use, simple to operate, stable in performance and highly sensitive.

technical background

Adenine nucleotides, adenosine, including adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate, are monomeric units that are components of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). And provide chemical energy to maintain the biochemical and physiological functions of tissue cell structures such as muscles and membranes in the energy metabolism pathway, regulate cell glycolysis, tricarboxylic acid cycle, electron transport system, and oxidative phosphorylation activities. Adenine nucleotides are low in concentration and unstable in cells, and their metabolism and energy status are usually evaluated by their different types, concentration differences, and distribution. Adenosine diphosphate (ADP) participates in the ADP-ATP cycle and provides thermal energy conversion and homeostasis, especially in mitochondrial aerobic respiration and photosynthetic synthesis. Adenosine triphosphate (ATP) is present in all metabolically active cells and is a hallmark of cell viability. Once the cells are apoptotic or necrotic, the ATP concentration drops sharply. The ADP/ATP ratio (ADP/ATP ratio) is often used to distinguish between cell death and survival: reproduction, apoptosis, and necrosis. It is widely used in cell viability and drug discovery research. Firefly luciferase is a 62 kd monomeric protein that catalyzes the oxidation of ATP-dependent luciferin, which converts chemical energy into oxidative oxyluciferin by electron transfer, and ATP. The quantities are linearly positively correlated. First, the ATP concentration was quantified by bioluminescence (560 nm) detection, and then ADP was converted to ATP in the presence of pyruvate kinase (PK), and then detected by a bioluminescence instrument to quantify the ADP concentration. ADP/ATP ratio. The way of reaction is:

product content

Cleaning solution (Reagent A) ml

Lysate (Reagent B) ml

Buffer (Reagent C) ml

Reaction solution (Reagent D) microliter

Conversion solution (Reagent E)

Product manual 1 copy

storage method

Save the cleaning solution (Reagent A) in the refrigerator at 4 °C; the rest is stored in the refrigerator at -20 °C, strictly avoiding the light and repeated freezing and thawing; effective guarantee for March

User-supplied

1.5 ml centrifuge tube: container for sample storage

15 ml conical centrifuge tube: container for sample processing

Black 96-well plate: container for cold light detection operations

(Micro) Benchtop Centrifuge: for sample handling

Cold light meter: for cold light detection of samples

Experimental procedure

• Sample preparation to be tested

• Surgical removal of animal tissue and weighing to determine 500 mg tissue weight
• (selection step) into a pre-cooled 15 ml conical tube
• (Selection step) Add 1x cleaning solution (Reagent A)
• Move into a liquid nitrogen cryotube
• Immediately put in a liquid nitrogen tank overnight
• The next day, it was taken out from the liquid nitrogen tank, and the tissue was ground into a powder (immediately) with a grinding rod (note: do not freeze the tissue )
• Put in a 15 ml conical tube
• Add xx microliters of lysate (Reagent B) in an ice trough
• Strong vortex for 30 seconds, mix thoroughly
• Incubate in the ice trough for 30 minutes, during which the vortex is vortexed for 30 seconds every 10 minutes (Note: If you need to temporarily stop, put it in the -70 °C refrigerator for storage )
• Immediately centrifuge in a 4°C benchtop centrifuge for 10 minutes at a speed of 10,000g
• Carefully remove the supernatant into a new sterile 1.5 ml centrifuge tube
• Store in a refrigerator at -70 ° C or in an ice bath

• Preparation for measurement

• Set the luminometer: temperature 25 ° C, 1 second delay (Delay), 1 second integration (Integration) detection. At the same time, close the light in the operating room
• Before the start of the measurement, the buffer (Reagent C) in the kit at -20 °C in the refrigerator was placed in an ice bath and thawed, then 500 μl was removed to a new 1.5 ml centrifuge tube, and xx μl of the reaction solution was added (Reagent D). ) , gently mix, placed in the ice trough, marked as the reaction working fluid , placed in the dark room ice tank for use. Then do the following.

• Activity assay

• Prepare a black 96-well plate
• Add 50 μl of sample to be tested
• Add xx microliters of reaction working solution
• Incubate for 3 minutes at room temperature (25 ° C) to avoid light
• Immediately put into the luminometer detection (1 second delay, 1 second detection): Obtain ATP Relative Light Unit (RLU)
• Allow to stand at room temperature (25 ° C) for 10 minutes to avoid light
• Immediately put into the luminometer detection (1 second delay, 1 second detection): Obtain ADP background Relative Light Unit (RLU)
• Add xx microliters of transformant (Reagent E )
• Incubate for 3 minutes at room temperature (25 ° C) to avoid light
• Immediately put into the luminometer detection (1 second delay, 1 second detection): Obtain ADP Relative Light Unit (RLU)
• Calculate sample ADP/ATP ratio

ADP/ATP ratio = (ADP relative illuminating unit - ADP background relative illuminating unit) ÷ ATP relative illuminating unit

• Result analysis (reference)

Precautions

• This product is 50 operations
• All operations must be performed aseptically
• Wear gloves when handling
• It is recommended to arrange 3 holes for each sample, that is, repeat 3 times, and take the average value when calculating.
• The reaction solution (Reagent D) is highly sensitive to light, so the whole operation must be carried out in the dark to avoid repeated freezing and thawing.
• Because ATP is ubiquitous and thermally stable. The recommended tips and utensils must be strictly sterile to avoid repeated use; avoid contaminating the reagent solution during operation, and do not touch the inner lid of the reagent container by hand.
• If the user does not have a luminometer, use a scintillation counter (scitillation counter or beta counter) instead, but with lower sensitivity
• If it is an injection luminometer, it is recommended to use a non-ionized water to rinse the luminometer injector before and after the test.
• The company provides a series of ATP detection reagent products

Quality Standard

• This product has been certified to be stable.
• This product has been identified and sensitive

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