Bbd 6220

Immortalised RAW264.7 macrophages (400319, CLS Cell Lines Service) were cultured in Dulbecco's modified Eagle's medium-high glucose (DMEM, D5671, Sigma-Aldrich), enriched with 10% fetal calf serum (FCS, P30-3302, PAN Biotech), 1% L-glutamine (G7513, Sigma-Aldrich), and 1% Antibiotic/Antimycotic Solution 100x (A5955, Sigma-Aldrich). The medium was replaced every two to three days. Cultivation was performed in an incubator (BBD 6220, Thermo Fisher Scientific) at 37°C and 5% CO₂ saturation. All cell culture experiments were carried out under sterile conditions (laminar flow unit, BDK Luft- und Reinraumtechnik).

Human choroidal vascular endothelial cells (HCVECS; #36052-03, Celprogen, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/l glucose supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The human macrophages were derived from human peripheral blood mononuclear cells and cultured, as the previous description^{22 (link)}. The cells were kept at 37 ^°C in a humidified atmosphere containing 5% CO₂. HCVECs were cultured in the lower well and macrophages were cultured in the upper well of the transwell plate (#CLS3397, Corning, USA) and verified by STR profiling.
The cells were culture in CO₂ incubator (#BBD6220, Thermo Fisher Scientific; 1%O₂, 94%N₂, and 5%CO₂) for 24 h (hypoxia group), HMPL-013 (0.05 μmol/l for 24 h), CCL2-neutralizing antibody (#AB-479-NA, R&D Systems, USA; 5 μg/ml for the last 30 min), recombinant human CCL2 protein (#279-MC, R&D Systems; 100 ng/ml for 24 h), geraniin (macrophage polarization modulator; #PHL80994, Merck; 30 μM for the last 2 h), or LPS (macrophage M1-type polarization agonist; Escherichia coli LPS, serotype 0127:B8; #L3129; Merck, USA; 2 μg/ml for 24 h).

3T3-L1 preadipocytes obtained from China Center of Type Culture Collection (CCTCC, Wuhan, China) were cultured in DMEM supplemented with 10% FBS, 100 g/mL streptomycin, 100 U/mL penicillin, 44 mM NaHCO₃ and 1 mM sodium pyruvate at 37°C in a 5% CO₂ humidification atmosphere (Thermo Scientific BBD6220, Germany). To induce differentiation, the cultured 3T3-L1 preadipocytes were kept until confluence was reached (0 day), and the culture medium was then replaced with a fresh induction medium containing 0.5 mM IBMX, 10 μg/mL insulin and 1 μM DEX in DMEM with 10% FBS for 2 days. The cells was then cultivated in a differentiation DMEM medium containing 10 μg/mL insulin and 10% FBS for four times (2 days for each time) until the cells were harvested.
The harvested cells were cultured in a complete medium containing 10 ng/mL TNF-α for 24 h (except the control group), then the media were removed and adherent cells were washed with PBS for twice. The obtained adipocytes were incubated in complete DMEM containing catechin (0, 10, 25, 50, 100 μg/mL) up to 48 h. Finally the adipocytes were collected for following experiments.