2.5. Human Microvascular Endothelial Cells (HMEC-1) Treatments and Viability

The human microvascular endothelial cell (HMEC-1) line was obtained from the Centers for Disease Control and Prevention (Atlanta, GA, USA) and grown as previously reported [18]. A toxicity curve using increasing concentrations of Castanea bee pollen (1, 10, 100, and 200 µg/mL) and thapsigargin (0.01–3 μM) was performed. Further experiments were carried out on HMEC-1 cells stimulated for 2 h with or without 0.3 μM thapsigargin, following 1 h pretreatment with increasing concentrations of Castanea bee pollen (0–200 µg/mL). The cell viability at all treatment conditions was evaluated by the MTT assay as previously described [19]. The optical density was recorded at 540 nm through a multiplate reader (Multiskan EX, THERMO, Waltham, MA, U.S.) and reflected the amount of metabolically active cells.

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