Atp determination kit a22066

For [NADH]/[NAD⁺] determination, cold methanol quenching was applied. Previous reports on actinomycetes suggest leakage caused by cold shock (Wittmann et al., 2004 (link)). Each sample with 50 μL cell culture was quenched by immediate addition of 500 μL methanol precooled at −80°C. 500 μL 4°C water and 500 μL −20°C chloroform were instantly added to samples. Samples was vortex vigorously and then stored at −20°C for 1 h. Supernatant was reserved by centrifugation at 10000 × g at 4°C for 10 min. NAD/NADH-Glo^TM assay kit (Promega, United States) was used for NADH/NAD⁺ measurement. For [ATP]/[ADP] ratio determination, cells were quenched by phenol as described previously (Michel et al., 2015 (link)). The amount of ATP or ADP was quantified with ATP determination Kit (A22066) (Invitrogen, United States). ADP was assayed after ATP had been determined by adding pyruvate kinase and recording the increase in luminescence. The results were corrected for quenching of the signal by the addition of pyruvate kinase.
The P value of their significance was calculated using Student’s t test.

ATP determination was performed using the Invitrogen ATP determination kit (A22066). MMP analysis was performed using a JC-1 (excitation filter 530/25, emission filter 590/35) MMP detection kit (Biotium). Cellular ROS generation was analyzed using 2,7-dichloro dihydrofluorescein diacetate (excitation filter 485/20, emission filter528/20) from the Invitrogen ROS detection kit. For all three analyses of mitochondrial stress, the reagents and reaction mixture were combined according to the supplied protocol. All fluorescence measurements were read using a Biotek Synergy H1 microplate reader.

For the measurement of intracellular ATP concentration, A549, HT-29, MRC-5, and U-937 cells were seeded on 6-well plates (TPP, Trasadingen, Switzerland) (0.5 × 10⁶ cells/well) and incubated under standard conditions for 24 h. Subsequently, 200 μg/mL XOS were added to duplicate wells of each cell line. Duplicate wells with cells cultured for the same time in complete DMEM served as positive control. At the end of the 24 h treatment, A549, HT-29, and MRC-5 cells were detached, and U-937 cells were resuspended. Then all cell samples were transferred to Eppendorf tubes, washed with sterile DPBS, and lysed. A negative control with nonlysed cells was set for each cell type. The ATP concentration was measured using a standard ATP Determination Kit A22066 (Invitrogen^TM, Thermo Fisher Scientific Inc., Waltham, MA, USA)) according to the manufacturer’s protocol. Detection of ATP was based on a bioluminescent reaction with D-luciferin catalyzed by the enzyme luciferase [65 (link)]. The reaction is extremely sensitive and allows the detection of ATP in a concentration of up to 0.1 picomole. Bioluminescence measurements were carried out on a GloMax^® 20/20 luminometer (Promega Corporation, Madison, WI, USA).

ATP concentration was measured in the hippocampal lysates using a luciferin/luciferase bioluminescence assay kit (ATP determination kit #A22066, Molecular Probes, Thermo Fisher Scientific, United States), as previously described (Jara et al., 2018 (link)). The amount of ATP in each sample was calculated from standard curves and normalized to the total protein concentration.

The levels of ATP and UDP in the culture supernatant of P. gingivalis, S. mitis, E. coli, and the supernatant of MG6 microglial cells after infection with bacteria were detected using the ATP Determination Kit A22066 (Molecular Probes Inc.) and MicroMolar UDP Assay Kits (ProFoldin Inc., Hudson, MA, USA), respectively, in accordance with the manufacturer’s protocols. The bacterial supernatants were obtained by centrifugation at 6,000× g for 20 min at 4 °C and filter-sterilized using a 0.2 μm filter for the assay. ATP levels were measured by detecting the luminescence using a Berthold Lumat3 LB9508 (Berthold Technologies, Oak Ridge, TN, USA), and the UDP levels were measured by detecting the fluorescence using an EnSight multimode plate reader (PerkinElmer, Waltham, MA, USA).