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HA, HBMEC, and HP cells (1 x [10.sup.4]) were seeded in each well of a 96-well cultured plate for 24 h and then treated with TMZ (200 [micro]M), PG (50 or 100 [micro]M), or their combination for 48 h.
Shim, "Oxidative stress induced by cigarette smoke extracts in human brain cells (T98G) and human brain microvascular endothelial cells (HBMEC) in mono- and co-culture," Journal of Toxicology and Environmental Health.
Abbreviations HBMEC human brain microvascular endothelial cell line RASMC rat aortic smooth muscle cell SMCs smooth muscle cells OGD oxygen and glucose deprivation EPCs endothelial progenitor cells FBS fetal bovine serum ARTICLE INFO
For in vitro proliferation assays, HBMECs were seeded into 96-well (1 x [10.sup.4] cells/well) flat bottom plates with medium alone (control) or medium containing different concentrations of astaxanthin (3,10 and 30 [micro]M).
HBMECs and RASMCs were seeded in 6-well plates (5 x [10.sup.4] cells/well) until the cells were fused to more than 90%, and discarded the culture liquid, then washed twice with PBS, added RPMI 1640 medium diluted with 3, 10 and 30 [micro]M astaxanthin in HBMECs, and DMEM medium diluted with 1, 3 and 10 [micro]M of astaxanthin in RASMCs, respectively, then used 200 [micro]l pipette tip to scratch and pictured after 24 h, and 48 h, measured the distance and counted in five random fields (x 100).
HBMECs (3 x [10.sup.4] cells/well) containing different concentrations of astaxanthin (3, 10 and 30 [micro]M), and RASMCs (2 x [10.sup.4] cells/well) containing different concentrations of astaxanthin (1, 3 and 10 [micro]M) were seeded in 24-well plates in serum-free media previously coated with growth factor-reduced matrigel matrix (BD Bioscience, San Jose, CA, USA).
To mimic the oxygen and glucose deprivation in vitro, HBMECs were incubated in a hypoxia solution for 6 h.
HBMECs were incubated with or without astaxanthin in a hypoxia solution for 6 h and HBMECs viability was assessed using an MTT assay.
HBMECs were cultured for 24 h, and RASMCs were cultured for 48 h, then washed twice with ice cold PBS on ice and lysed in NP40 lysis buffer (Biosource, Camarillo, CA, USA) (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM [Na.sub.3]V[O.sub.4], 1% NP-40 and 0.02% Na[N.sub.3]) supplemented with 1 mM PMSF and 1 x protease inhibitor cocktail (Sigma, Saint Louis, MO, USA).
Astaxanthin augments proliferation, migration and tube formation HBMECs were incubated with different concentrations of astaxanthin (3-30 [micro]M) for 24 h and RASMCs were incubated with different concentrations of astaxanthin (1-10 [micro]M) for 48 h, respectively.
Effects of astaxanthin on cultured HBMECs against hypoxia-induced cytotoxicity
However, HBMECs were incubated with astaxanthin; cell viability was significantly increased in a concentration-dependent manner.
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