ATP Assay Kit

Reagent Preparation

1. 1× ATP lysis solution: dilute ATP Detection Reagent C (10×) 1:10 with sterile deionized water, mix, and keep on ice.

2. ATP detection diluent: dilute ATP Detection Reagent B (10×) 1:10 with sterile deionized water.

3. ATP detection working solution: for each 1 mL ATP detection diluent, add 10 μL ATP Detection Reagent A (100×), 10 μL ATP Detection Reagent D (100×), and 50 μL ATP Detection Solution (20×). Mix gently and keep on ice protected from light. Prepare only the amount required for the assay.

4. ATP standards: serially dilute the 0.5 mM ATP Standard Solution with 1× ATP lysis solution to 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 once with PBS. Add 200 μL pre-cooled 1× ATP lysis solution per well of a 6-well plate, lyse thoroughly, and collect the lysate.

2. Suspension cells: collect the cells, centrifuge at 600 × g for 5 min, and discard the supernatant. Add 100-200 μL pre-cooled 1× ATP lysis solution per 1 × 10⁶ cells and lyse thoroughly.

3. Tissue: weigh 10-30 mg tissue, add 100 μL pre-cooled 1× ATP lysis solution per 10 mg tissue, and homogenize thoroughly on ice.

4. Centrifuge cell or tissue lysates at 12,000 × g for 5 min at 4°C. Keep the supernatant on ice for the assay.

Assay Procedure

1. Equilibrate the ATP detection working solution and samples to the assay temperature. Add 100 μL ATP detection working solution to each well of a white opaque 96-well plate.

2. Allow the plate to stand at room temperature for 3-5 min until the background luminescence stabilizes.

3. Add 20 μL ATP standard or sample supernatant to each well and mix rapidly. Two or three replicate wells are recommended.

4. Immediately measure luminescence using a luminometer or luminescence microplate reader. An integration time of 0.5-1 s may be used, and identical settings should be applied to all wells.

Calculation

Plot the ATP standard concentration against blank-corrected luminescence to generate a standard curve and determine ATP concentrations in samples. Multiply by the dilution factor and normalize to cell number, tissue weight, or protein content as required.