Phosphate buffered saline (pbs)

The migratory ability of the cells was assessed using a scratch-wound directional assay. Briefly, the Ea.hy926 cells were seeded at a cell density of 8×10⁴ cells/well in a 24-well cell plate (Corning, Inc.), and grown overnight into a confluent monolayer. A sterile 20–200 μl micropipette tip (Axygen^®; Corning Life Sciences, Tewksbury, MA, USA) was then used to create a ‘wound field’ of ±1000 μm width. The cells were washed twice with phosphate-buffered saline (Boster, Wuhan, China) and replaced in fresh medium containing the indicated concentrations of TSAJ, supplemented with 0.5% FBS and VEGF (10 ng/ml).
Following a 14 h incubation, the migrated cells were photographed using an inverted microscope (TE2000-S; Nikon Corporation, Tokyo, Japan) with NIS-Elements software. Cell migration was estimated by measuring the endothelial cells that had migrated from the edge of the wounded monolayer (23 (link)). The percentage of migration was the mean calculated from five replicates of each experiment. Three independent experiments were performed. The control group, which did not receive VEGF or TSAJ treatment, was set at 100%.

Primary microglia were isolated from brains of neonatal C57BL/6 mice (P0–P3) as described.²¹ The purified microglial cells were cultured at 37°C in DMED–F12 medium (Gibco) containing 10% fetal bovine serum (Gibco). After 7 days, microglia were pre‐treated with 10, 50, or 100 μM ASD (Alfabiotech) or pioglitazone (10 μM, Sigma‐Aldrich).³⁷ After 30 min, microglia were treated for 24 or 48 h with either 50 ng/mL LPS (Sigma‐Aldrich) or phosphate‐buffered saline (PBS; BOSTER). Experimental groups were as follows: control group (Ctrl), not treated with ASD or LPS; LPS group (LPS), treated with LPS but not ASD; LPS + ASD (10, 50, 100 μM) group, treated with LPS and ASD at the indicated concentrations; and LPS + pioglitazone group, treated with LPS and 10 μM pioglitazone. At each time point, microglia were collected and transferred to new plates for further experiments.

All oligonucleotides were obtained from Sangon Biotech (China). Magnesium chloride hexahydrate was purchased from Tian Li (China). Trizma base was obtained from Sigma-Aldrich (USA). Agarose was purchased from Lonza (USA). DNase I was obtained from Solarbio (China). Lipofectamine 2000 (LP2000) was purchased from Invitrogen (USA). FM4-64 was purchased from Molecular Probes (USA). Phosphate-buffered saline (PBS) and Mueller–Hinton broth (MHB) were purchased from Boster (China) and Land Bridge (China), respectively. The PrimeScript RT Reagent Kit with DNA Eraser and Premix Taq RT-PCR System were from Takara Bio Inc (Japan). Thse water used in all experiments was prepared via a Millipore Milli-Q purification system with a resistivity greater than 18 MΩ cm⁻¹.

Mice were arbitrarily divided into four groups: (1) a Sham group (n = 10): animals received femoral artery catheterizations without drawing blood; (2) a hemorrhagic shock (HS) group (n = 10): animals underwent HS and received an i.p. injection of 100-μL phosphate-buffered saline (PBS) (Boster, Wuhan, China) at the end of the 90-min shock period; (3) a kaempferol pretreatment (Kae PT) group (n = 10): animals received an injection of kaempferol (Sigma, St. Louis, MO, USA) (i.p., 10 mg/kg BW, dissolved in 100 μL of PBS) 12 h prior to the initiation of HS; and (4) a kaempferol treatment (Kae T) group (n = 10): HS animals received a kaempferol injection (i.p., 10 mg/kg BW in 100 μL of PBS) at the end of the shock period (Figure 1).Figure 1

Study protocol. Animals were assigned to Sham (n = 10), HS (n = 10), Kae T (n = 10), and Kae PT (n = 10) groups. The HS group received an injection of 100-μL PBS (i.p.) at the end of the shock period. Kae T and Kae PT groups received an injection of kaempferol (i.p., 10 mg/kg BW, dissolved in 100-μL PBS) 90 min following or 12 h prior to the initiation of HS, respectively. Animals were sacrificed 6 h after the initiation of HS for blood and tissue sample collection.

Doxorubicin hydrochloride (Dox·HCl) was purchased from Meilunbio (Dalian, China). Polyethyleneimine (PEI, average Mn = 25,000) and DMEM/F-12 containing l-glutamine were purchased from Sigma. Fetal bovine serum (FBS), DMEM/F12 (1:1) cell culture media, 0.05% trypsin-EDTA, penicillin-streptomycin solution, and other cell culture-related reagents were purchased from Gibco (Carlsbad, USA). Hoechst 33,342, Dio, cell counting kit-8 (CCK-8), enhanced BCA protein assay (BCA) kit, and RIPA lysis buffer were purchased from Beyotime Biotechnology Co. (Shanghai, China). Phosphate-buffered saline (PBS) was purchased from Boster Biological Technology (Pleasanton, CA, USA). Polyethylene terephthalate (PET) was used as the experimental substrate in material characterization and in vitro experiments. A serial of antibodies used for western blotting (WB) were purchased from Santa Cruz. Foldable hydrophobic acrylic IOLs were supplied by 66 Vision Technology Co., Ltd. (Suzhou, China).