Atp bioluminescence assay kit hs 2

Urinary ATP levels (mol/ml) were estimated by luminometry using the ATP Bioluminescence Assay Kit HSII (Sigma, Portugal). Luciferase was added to the well, followed by an equal volume of sample. The highest value of luminescence was registered for each sample. After stabilization of the lowest luminescence reading, standard addition was performed to prevent erroneous outcomes due to any urinary compound interference.

A mixture of luciferin-luciferase was added according to the manufacturer instructions using the ATP Bioluminescence Assay Kit HS II (adenosine triphosphate (ATP) Bioluminescent Assay Kit, Sigma-Aldrich, St. Louis, MO, USA) as previously described [31 (link)]. ATP detection was evaluated using a multimode microplate reader (Synergy HT, BioTek Instruments Inc., Vermont, USA) controlled with Gen5 Data Analysis Software (BioTek). Sample bioluminescence was compared to that of standard amounts of ATP used in the same concentration range; standard ATP samples were prepared daily. All samples were run in duplicate. Cystometric void data were obtained from at least three voids/animal from control (n = 6 each) and all experimental groups (n = 6 each) and urinary bladder data were obtained from control (n = 6 each) and all experimental groups (n = 6 each). Vehicle for Tempol did not affect the ATP determinations. The ATP in cystometric fluid or ATP content in urinary bladder was calculated relative to the standard curve and expressed as nmol per total protein or nmol per mL of urine.

Whole-cell ATP levels were monitored using the ATP Bioluminescence Assay Kit HSII according to manufacturer instructions (Sigma-Aldrich S.A.R.L.). ATP levels were normalized to the total amount of proteins dosed with Bradford assay against BSA standards (Thermo Fisher Scientific).

Cells were collected into pre-cooled PBS, frozen quickly as aliquots, and stored in liquid nitrogen. For use, aliquots were allowed to melt slowly in an ice water bath and vortexed for 10 s. The ATP content was assayed via Luciferase driven bioluminescence using an ATP Bioluminescence Assay Kit HS II (Sigma-Aldrich, 11699709001). The relative light units of each sample were detected with Microplate Reader (M5; Molecular Devices, San Jose, CA, United States).

Around 20,000 cells per well under conditions of scramble and knockdown were plated in 96-well plates one day before the assay. Next day, the amount of total ATP was determined using the ATP Bioluminescence Assay Kit HS II (Sigma catalogue number 11699709001) and analyzed on an Infinite M200 PRO Luminometer (Tecan). Values obtained from the Infinite M200 PRO Luminometer were further normalized by the protein amount under each condition.