Low dose Met attenuates Dox induced cytotoxicity in H9c2 rat cardiomyoblasts.

<p>(A) H9c2 rat immortalized cardiomyoblasts were cultured with reduced FBS (1%) for up to 72 hours in 96-well culture plates under indicated conditions. Viability levels were estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104888#s2" target="_blank">Materials and Methods</a>. (B) LDH leakages in H9c2 cells after Dox/Met treatment. Cells were treated with indicated conditions for 24 hours in serum free medium. LDH levels in culture supernatants were evaluated using LDH assay kit. (C) H9c2 cells were cultured on poly-L-lysine coated chamber slides for 24 hours under the conditions as indicated in reduced FBS medium. The slides were immunostained with anti-Ki-67 antibody (BD Biosciences) and ALP conjugated secondary antibody (Vector Laboratory). The numbers of Ki-67-positive vessels were counted in six randomized fields of the chambers. The total number of cells from each group was counted and normalized to each chamber. (D) H9c2 cells were cultured with conditions and for hours as indicated in serum free medium. Cellular ROS generations were measured using a detection assay kit. (E) H9c2 cells were cultured with serum free medium under the indicated conditions. Intra-cellular calcium levels ([Ca²⁺]_i) were measured using fluo-4 (Molecular Probe), a fluorescent Ca²⁺-indicator dye. Values represent mean ± S.D. (n = 4) from quadruplicate samples for each treatment at varying treatment conditions. ^* p<0.05; ^# p<0.05 vs CTR; ^$ p<0.05 vs Dox 10 nM alone.