Sirt3 activity assay kit

The mitochondrial isolation from fresh hippocampus tissues was performed using the mitochondrial isolation kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). According to the manufacturer’s instructions, the hippocampus tissues were homogenized using glass homogenizer within mitochondrial isolation buffer, then centrifuged at 700×g for 10 min at 4°C, and the supernatant was centrifuged at 3000×g for 15 min. The pellets were used for MMP and Sirt3 deacetylation activity freshly.
The MMP was determined by the JC-1 MMP detection kit (Biotium, Hayword, CA, U.S.A.), as to the methods previously described in other study [20 (link)] and the manufacturer’s instructions. The Sirt3 deacetylation activity was measured using the Sirt3 activity assay kit (#ab156067, Abcam, Cambridge, MA), following the protocols provided by the manufacturer and method previously described in other study [8 (link)].

SIRT3 Activity Assay Kit (Abcam, USA) was used according to the manufacturer’s instructions. The reaction mixture contains assay buffer, Fluoro-Substrate peptide, NAD, developer, and samples. Read fluorescence intensity for 30 min to 60 min at 1 min to 2 min intervals using microtiter plate fluorometer with excitation at 340–360 nm and emission at 440–460 nm. Measure and calculate the rate of reaction while the reaction velocity remains constant.

To characterize deacetylation activity of recombinant, N-terminal truncated elephant shark SIRT3.2 (ΔNT-SIRT3.2), we used a fluorometric SIRT3 activity assay kit (Abcam, cat # ab156067; Cambridge, UK), according to the manufacturer's instructions. This assay allows detection of a fluorescent signal upon deacetylation of an acetylated substrate peptide for recombinant human SIRT3. The intensity of fluorescence was measured on a fluorometric microplate reader (Varioskan Flash, ThermoFisher) with excitation set at 350 nm and emission detection set at 450 nm. To analyze the deacetylation activity of N-terminal truncated elephant shark SIRT3.2 protein, first we determined its optimum temperature that resulted to be ∼24 °C. Subsequent analyses were performed by comparing the activity of N-terminal truncated elephant shark SIRT3.2 protein at 24 °C to that of human SIRT3, which is provided by the assay kit, at 37 °C. K_m and V_max were obtained by varying the concentration of the fluorogenic acetylated substrate peptide provided by the assay kit. This assay was also used to determine the effect of NAM, QUE, and resveratrol (REV; Sigma-Aldrich) on the deacetylation activity of ΔNT-SIRT3.2.

BMMs were isolated as described above and stimulated with RANKL to form preosteoclasts on 6-well plates. Cell pellets were washed with cold PBS and treated with mitochondria isolation reagents supplied in the kit. The cell lysates were then kept on ice for 5 min and centrifuged at 700 g for 10 min at 4 °C. The supernatant was transferred to new tubes and centrifuged at 12,000 g for 5 min at 4 °C to isolate the mitochondrial potion, after adding 2% CHAPS in Tris to the pellets. The protein concentration of cell lysates was determined using a DC Protein Assay kit (Bio-Rad). Sirt3 activity was measured using a Sirt3 Activity Assay Kit (Abcam, Cambridge, United Kingdom) according to the manufacturer’s directions. The released fluorescent signal was measured in a microplate fluorescence reader with excitation/emission wavelengths of 340/440 nm.

Sirt3 deacetylase activity was determined by using SIRT3 Activity Assay Kit (ab156067, Abcam, Cambridge, MA, USA) according to the manufacturer’s instruction. NRK-49F cells were treated with TGF-β1 and/or PAA for 12 h and 24 h. Sirt3 activity was measured by detecting fluorescent emission at 460 nm and following excitation at 360 nm.

The activity of SIRT3 was measured by means of a fluorometric method using the SIRT3 Activity Assay Kit (Abcam, Cambridge, UK). Three types of blanks were analyzed: one without patient sample, one without enzyme recombinant, and one without NAD⁺, and the respective volumes were substituted with double-distilled water. The standard curve for SIRT3 expression in PBMCs was fitted using analyses of 0.5, 1.0, 1.5, and 2.0 μg of recombinant SIRT3. Deacetylase activity of SIRT3 was evaluated using kinetic measurements at 2 min intervals using a microtiter plate fluorometer (FLx800, BioTek Instruments, USA) with an excitation set at 340–360 nm and emission at 440–460 nm during 30 min. The rate of reaction was calculated while the reaction velocity remained constant. The expression of SIRT3 was evaluated by means of end-point measurements based on the calibration curve using recombinant SIRT3 (Abcam, Cambridge, UK). The expression of SIRT3 in PBMCs was represented as μg/mg of proteins and deacetylase activity as units (U)/mg of proteins.

After mitochondria were isolated from cells or brain tissues, Sirt3 deacetylation activity was measured using Sirt3 activity assay kit (#ab156067, Abcam, Cambridge, MA) following the manufacturer’s protocol. The fluorescent intensity was collected on Tecan infinity M200 pro according to the manufacturer’s protocol and was normalized with the amount of total test protein. Final data were normalized with total protein concentration from cells or tissues.
Sirt3 deacetylation activity was represented as the ratio of fluorescent intensity to the amount of total test protein and the ratio was used for statistical analysis.