ATP assay Sample Clauses

ATP assay. ATP content was assessed using the ATP-Glo kit (Biotium, California, USA) as previously described (Xxxxxxx et al. 2011). Briefly, ATP-Glo cocktail mix was made by dissolving 10 mg of D-luciferin in 1 mL of deionised water (final concentration 10 mg/mL). Eight hundred µL of D-luciferin solution was then added to 20 mL ATP assay buffer (final concentration 0.4 mg/mL D-luciferin). Two hundred µL of firefly luciferase was added to complete the cocktail. After dosing as described in Section 2.4.2, plates were transferred without removal of media to a spectrophotometer equipped with an injector (Fluostar Omega, BMG Labtech, Buckinghamshire, UK). The spectrophotometer was programmed with the following protocol: injection of 100 µL of ATP-Glo cocktail, shake for 3 seconds, delay for 10 seconds and then measure luminescence for 10 seconds using a gain of 2000. All xxxxx were sequentially injected and measured individually. Results were calculated and expressed as described in section 2.4.4. In the event that absolute ATP values were to be determined via a calibration curve, a set of standards were made as follows: • 10,000 pmol ATP: 5 µL of a 2 mM ATP solution was added to 995 µL of PBS • 1000 pmol ATP: 100 µL of the above solution was added to 900 µL of PBS • 100 pmol ATP: 100 µL of the above solution was added to 900 µL of PBS • 10 pmol ATP: 100 µL of the above solution was added to 900 µL of PBS • 1 pmol ATP: 100 µL of the above solution was added to 900 µL of PBS • 0.1 pmol ATP: 100 µL of the above solution was added to 900 µL of PBS • 0.01 pmol ATP: 100 µL of the above solution was added to 900 µL of PBS One hundred µL of each standard were then loaded in triplicate onto the same white 96-well plate as the relevant experimental samples before the assay was performed as described above. Taking into account the addition of 1/10th of the stock solution of the standards into each well, the final amounts of ATP in each triplicate set of xxxxx were 1000 pmol, 100 pmol, 10 pmol, 1 pmol, 0.1 pmol, 0.01 pmol, and 0.001 pmol. The calibration curve was constructed by creating a double log plot of luminescence against concentration of ATP. A line of best fit was then determined via linear regression.
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