4 5 diaminofluorescein diacetate daf 2da

Rapamycin, DAPI, 4,5-Diaminofluorescein diacetate (DAF-2DA), and dihydroethidium (DHE) were purchased from Sigma-Aldrich. MitoSOX Red, MitoTracker Red CMX ROS, and Trizol reagents were obtained from Invitrogen. Primary antibodies of phospho-mTOR (Ser2448), mTOR, phospho-p70 S6 (Thr389), p70 S6 Kinase, phospho-Akt (Ser473), Akt, Rictor, Raptor, and secondary antibody were obtained from Cell Signaling Technology. Reverse transcription reagent Kit and SYBR real-time PCR kit were from Takara (Dalian, China). Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) kit was purchased from Roche Applied Science (Indianapolis, IN).

The drugs used in the present study include Fura2-AM, adenosine 5′ triphosphate (ATP), donepezil, acetylcholine, methyllycaconitine (MLA), BD1047, BD1063, LY294002, U73122, PD98059, KN-62, 4,5-diaminofluorescein diacetate (DAF-2DA), and human recombinant TNFα were purchased from Sigma-Aldrich (St. Louis, MO). MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] was purchased from Tocris Bioscience (Bristol, UK). human recombinant TNFα was diluted with the standard external solution to obtain the final concentration. donepezil was diluted with the standard external solution to obtain the final concentration (5 μM). This donepezil concentration is sufficient to inhibit the AChE activity in both human blood cells and monkey brain samples [23 (link)] or to suppress the LPS-induced NO production in mouse primary microglial cells [21 (link)]. Drugs that were insoluble in water were first dissolved in dimethylsulfoxide (DMSO; Wako Pure Chemical Industries, Osaka, Japan) and then diluted in the standard external solution. The final concentration of DMSO was always less than 0.1%.

HAoEC (PromoCell) were cultured in DMEM without phenol red containing l-glutamine (584 mg/liter) and fetal bovine serum (10%) (Thermo Fisher Scientific). The cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per well and allowed to grow for another 2 days, followed by serum fasting overnight and subsequent stimulation with ACh (10 μM) in the absence or presence of recombinant human IL-1β (10 ng/ml; Sigma-Aldrich) for 15 min. For measurement of intracellular NO, 4,5-diaminofluorescein diacetate (DAF-2DA; 10 μM) (Sigma-Aldrich) was added to the cells for the last 5 min in the dark (37°C, 5 min). Fluorescence images were captured. Alternatively, HAoEC were seeded in 96-well plates at a density of 0.25 × 10⁵ cells per well and grown for 2 days. DAF-2DA (10 μM) was added 5 min before harvesting, and the fluorescence intensity of the cells was measured using a spectrofluorometer with excitation wavelength at 495 nm and emission wavelength at 515 nm. The relative fluorescence intensity was normalized with protein concentration in cell lysate.