Aposensor adp atp ratio bioluminescent assay kit

The amount of ATP and ADP/ATP were measured by ApoSENSOR Cell Viability Assay Kit and ApoSENSOR ADP/ATP Ratio Bioluminescent Assay Kit (BioVision, California, USA) following the manufacturer's instructions. In brief, to measure intracellular ATP, 5 × 10⁴ GFP⁺ M1 cells were lysed with 250 μl lysis buffer on ice for 5 min. ATP monitor enzyme and lysis buffer were pre-mixed and the background luminescence value was designated as A. Cell lysate (50 μl) was added to the pre-mixed well, then the luminescence reading was designated as B. ADP converting enzyme was added to the well, then the luminescence reading was designated as C. The value of intracellular ATP is (B-A). The value of intracellular ADP/ATP is (C-B)/(B-A). The tests were done in duplicates.

An ApoSENSOR ADP/ATP Ratio Bioluminescent Assay kit (Biovision, San Francisco, CA, USA) was used to measure ADP/ATP ratio. This kit detects the ATP level based on bioluminescence via a luciferase catalyzed reaction. The ADP level is measured by its conversion to ATP catalyzed by ADP converting enzyme. We modified the manufacturer’s instructions and optimized the protocol to specifically measure the viability of hepatocytes. Briefly, 5 × 10⁴ hepatocytes were suspended in a single tube, then mixed with nucleotide releasing buffer (200 μl) for 10 min at room temperature. Thereafter, ATP monitoring enzyme (10 μl) was added to the solution, and the ATP level was measured using a GloMax-20/20 Luminometer (Promega, Madison, WI, USA) and expressed as the number of relative light units (RLU). After 10 min, the ADP in the solution was converted to the ATP by adding of ADP converting enzyme (10 μl), and the RLU was measured immediately before and after 5 min of conversion. Subsequently, the ADP/ATP ratio of the hepatocytes was calculated as (C-B)/A, where A is the RLU for ATP, B is the RLU of ATP immediately before conversion from ADP, and C is the RLU of ATP at 5 min after conversion from ADP; the evaluation was completed in 25 min.

On the day of the assay, the islets were placed in HBSS containing 1.1 mM glucose for 2 h. Then, 15 similar-sized islets/well were hand-picked and treated with 3.3 mM glucose or 22.2 mM glucose. After treatment for 2 h, the ATP/ADP ratio was measured using ApoSENSOR ADP/ATP Ratio Bioluminescent Assay Kit (BioVision, Milpitas, CA, USA), per the manufacturer’s instructions.

ATP/ADP ratio was analyzed using ApoSENSOR™ ADP/ATP Ratio Bioluminescent Assay Kit (BioVision). Briefly, 100 μl reaction mix in a 96-well plate was read as the background luminescence (Data A). After treatment, the cells were incubated with 50 μl nucleotide releasing buffer for 5 min. The lysate was transferred into the above 96-well plate, and 2 min later, the signals were read as Data B. Then, the samples were read again to determine the ADP levels (Data C). Finally, 1 μl ADP converting enzyme was added to the reaction to determine Data D. ATP/ADP Ratio was calculated using formula: (Data B − Data A)/(Data D − Data C). The ADP/ATP ratio was normalized to protein concentrations.

The aldolase activity was determined by Aldolase Activity Colorimetric Assay Kit (BioVision, USA) according to the manufacturer’s protocol. The Lactate Colorimetric Assay Kit II (BioVision) and the PicoProbeTM Fructose-1,6-Bisphosphate Assay Kit (BioVision) were applied to detect the levels of lactate and F1,6BP. The intercellular ATP levels were detected by ATP detection kit (Beyotime) and ADP/ATP ratio was determined using ApoSENSOR™ ADP/ATP Ratio Bioluminescent Assay Kit (BioVision) according to manufacturer’s instructions.

The ATP/ADP ratio in the hemocyte samples collected from the α-KG replenishment experiment was determined with an ApoSENSOR^™ ADP/ATP Ratio Bioluminescent Assay Kit (Biovision) using a slightly modified protocol as described in a previous study [4 (link)]. For each sample in a 96-well plate, 10 μl of hemocytes were mixed with 100 μl Nucleotide Releasing Buffer and shaken for 5 min. After adding 1 μl ATP Monitoring Enzyme to each well and allowing the reaction to proceed for 2 min, the luminescence (B) was determined by using a luminometer. After 10 min, the luminescence was recorded again to provide another data point (C). Next, to measure the ADP level in the cell, 1 μl ADP Converting Enzyme was added into each well and the luminescence (D) was measured again 2 min later. For the background control, 10 μl PBS was subjected to the same procedure to get the corresponding data point B’, C’ and D’. The ATP/ADP ratio was then calculated as: (B–B’)/([D–D’]–[C–C’]).

ADP/ATP ratios were measured from cultures (1x10⁶ conidia/mL) grown in liquid GMM at 37°C for 16 hours with shaking at 250rpm. Fungal tissue was washed with sterile water, and transferred to fresh GMM for 2 additional hours at 37°C with shaking at 250rpm. Tissue was lyophilized, ground with glass beads with a Mini-BeadBeater (BioSpec Products, Inc.). Metabolites were extracted using buffer from ApoSENSOR ADP/ATP Ratio Bioluminescent Assay Kit (K255-200, BioVision) and quantified following manufacturers protocol. Tissue lysates were filtered through Ultracel 10K centrifugal filters (Millipore) prior to processing to remove proteins.