Constant temperature incubator

The mouse hippocampal neuronal cells (HT22) (Sebacon Biotechnology, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, United States) in a constant temperature incubator (ThermoFisher Scientific, United States) at 37°C, 5% CO₂, and saturated humidity. Treatment was performed according to the following groups: control, BHBA (20 mM), D-gal (200 mM), D-gal + BHBA (5 mM), D-gal + BHBA (10 mM), D-gal + BHBA (20 mM), D-gal + donepezil (2 μM). BHBA, donepezil, D-galactose, and equal amounts of the blank solvent were applied separately according to the groups for 24 h, and the cells or culture medium were collected. All drugs were dissolved in serum-free DMEM.
In the experiments with SIRT1 inhibitor EX527, HT22 cells were divided into five groups: control, BHBA (20 mM), D-gal (200 mM), D-gal + BHBA (20 mM), and D-gal + BHBA (20 mM) + EX527(1 μM). EX527 was added 4 h before cell administration, followed by subsequent manipulations.
In the experiments with si-SIRT1 inhibition, HT22 cells were divided into five groups: control, BHBA (20 mM), D-gal (200 mM), D-gal + BHBA (20 mM), and D-gal + BHBA (20 mM) + si-SIRT1. si-SIRT1 (5′-3': GCA​CCG​AUC​CUC​GAA​CAA​UTT; 5′-3': AUU​GUU​CGA​GGA​UCG​GUG​CTT) was transfected with lipo3000 for 24 h, followed by subsequent operations.

A refrigerator was bought from Siemens (Germany). Spectra Max M microplate reader was obtained from Molecular Devices in the United States. A constant temperature incubator and Nunc‐Immuno 96‐well plates were supplied by Thermo Fisher (USA). A micropipette and CPA224S electronic analytical balance were purchased from Eppendorf (Germany) and Sartorius (Germany), respectively. Graduated flasks and beakers were from Tianjin Glass Instrument Factory (Tianjin, China). A vortex mixer was obtained from Beijing North TZ‐Biotech Development Co., Ltd. (Beijing, China).

TM3 cells were purchased from the National Experimental Cell Resource Sharing Platform (Beijing, China). Cells were cultured in a constant temperature incubator (Thermo Fisher Scientific, Massachusetts, USA) with 5% CO₂ at 37°C. Cell culture was performed in DMEM/F12 medium with 2.5% fetal bovine serum, 5% horse serum, and 1% penicillin–streptomycin solution.

RAW 264.7 cells were donated by Xinjiang Uygur Autonomous Region Institute of Medicine, Maintain the cells in DMEM/High Glucose medium (HyClone, Logan, Utah, United States) supplemented with 10% v/v fetal bovine serum (FBS, Gibco, United States) and 1% penicillin-streptomycin solution (HyClone, Logan, Utah, United States), and put them into 25 cm² culture flasks, and then place them in a constant temperature incubator (Thermo Fisher Scientific, United States) at 37°C and 5% CO₂, Subculture every 2–3 days to maintain logarithmic growth. DMSO (Beijing Solarbio Technology Co., BeiJing, China) was used to prepare Lactucin solutions of different concentrations and added to the cell culture fluid. The proportion of DMSO in the cell culture fluid was 0.1% (v/v).

HEFs were obtained from Chinese Academy of Medical Sciences, Institute of Basic Medicine and cultured in Low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM, Gibco, Grand Island, NE, USA) with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco, Grand Island, NE, USA). Constant temperature incubator (Thermo Fisher, Waltham, MA, USA) conditions were strictly controlled at 37 °C, 95% relative humidity, and 5% CO₂. HEFs were continuously passaged and stopped proliferating around 52 population doubling level (PDL). According to the age definition in cell culture, we divided them into young cells (22PDL), mid-aged (35PDL), and replicative senescence cells (49PDL).HEFs around 22PDL were exposed to 400 μmol/L H₂O₂ (BDH Chemicals Ltd., Poole, England) for 2 h every day for 4 consecutive days; the cells group was called the premature senescence initiation group (PSi) and then given fresh L-DMEM with fetal bovine serum and cultured for another 7 days, defined as the premature senescence persistence group (PSp), as previously described [20 (link)].