Gibco™ RPMI 1640 Medium

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	supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin

Protocol tips
supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin

Publication protocol

PAECs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI1640 medium (Gibco, Life Technologies, Rockville, MD, USA) according to the supplier instructions. The medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and cells were used at third passage for experiments.

The CCK-8 assay was used to evaluate PAEC proliferation using a CCK-8 cell proliferation assay kit (Beyotime, Shanghai, China) [34]. PAECs seeded in 96-well plates (104 cells/well) were pretreated with indicated concentrations of quercetin for 1 h and then stimulated with 100 ng/ml TGF-β1 for 48 h. Next, 10 μl CCK-8 solution was added to each well and incubated for 4 h. After the medium was removed, 100 μl dimethyl sulfoxide (DMSO) was added to each well. The culture plate was oscillated for 10 min in the shaking table to adequately dissolve the crystals. Finally, cell viability and proliferation were measured by reading the absorbance at 450 nm with Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA).

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