ATP Assay Kit

Reagent Preparation

1. 1× ATP Lysis Buffer: Dilute an appropriate amount of ATP Detection Reagent C (10×) 10-fold with 1× ATP Detection Buffer to prepare 1× ATP Lysis Buffer.

2. ATP Detection Buffer: Thaw the required reagents on ice in advance. Based on 100 μL ATP Detection Working Solution required for each sample or standard, dilute an appropriate amount of ATP Detection Buffer (20×) 20-fold with ultrapure water to prepare 1× ATP Detection Buffer. Keep the prepared buffer on ice.

3. ATP Detection Working Solution: Prepare the working solution by adding 40 μL ATP Detection Reagent A (100×), 40 μL ATP Detection Reagent D (100×), and 400 μL ATP Detection Solution B (10×) to every 3520 μL ATP Detection Buffer. Mix gently and keep on ice protected from light. Prepare fresh according to the actual number of wells and use immediately.

4. ATP Standard Solutions: Serially dilute the 0.5 mM ATP Standard Solution with 1× ATP Lysis Buffer to prepare ATP standards at 0, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM.

Sample Preparation

1. Adherent Cells: Remove the culture medium and wash the cells once with PBS. For cells cultured in a 6-well plate, add 150-200 μL of pre-chilled 1× ATP Lysis Buffer to each well. Lyse the cells thoroughly and collect the lysate.

2. Suspension Cells: Collect the cells and centrifuge at 600 × g for 5 min. Discard the supernatant. Add 200 μL of pre-chilled 1× ATP Lysis Buffer per 1 × 10⁶ cells and lyse thoroughly.

3. Tissue Samples: Weigh 10-30 mg of tissue. Add 100-200 μL of pre-chilled 1× ATP Lysis Buffer per 10 mg of tissue and homogenize thoroughly on ice.

4. Centrifuge the cell or tissue lysates at 12,000 × g for 5 min at 4°C. Collect the supernatant and keep it on ice for subsequent detection.

Detection Procedure

1. Equilibrate the ATP Detection Working Solution and samples to the temperature required for detection. Add 100 μL of ATP Detection Working Solution to each well of a white opaque 96-well plate.

2. Incubate at room temperature for 3-5 min to allow the background luminescence to stabilize.

3. Add 10 μL of ATP standard solution or sample supernatant to each well and mix immediately. It is recommended to test each sample in 2-3 replicate wells.

4. Immediately measure the luminescence using a chemiluminescence microplate reader. The integration time may be set to 0.5-1 s, and identical detection settings should be used for all wells.

Data Analysis

Plot the ATP standard concentration on the x-axis and the blank-subtracted luminescence values on the y-axis to generate a standard curve, and calculate the ATP concentration of each sample. Multiply the calculated value by the sample dilution factor, and normalize the results to cell number, tissue weight, or protein content as appropriate.