Quercetin q4951

EAC (Ehrlich ascites carcinoma cells) and human Breast cancer cell lines (MDA-MB-231 and BT-474) were kind gift from Dr. MVVST SubbaRao, CEMR laboratory. Quercetin (Q4951) and vitamin D3 (C9756) were procured from Sigma-Aldrich, United States. WST-1 reagent was procured from TAKARA. Fertilized chicken eggs were procured from Ilavala poultry farm, Mysuru. The Ehrlich ascites carcinoma (EAC) model is very commonly used model to study the pathological conditions of breast cancer and associated tumor angiogenesis. These EAC cells were first discovered and isolated by the Nobel Laureate, Paul Ehrlich in the mammary gland tumor of a white mouse, therefore these tumor cells were named after him to honor his great contribution to tumor biology. Recently, we further developed this model to study the breast cancer induced liver inflammation and fibrosis (9 (link)).

From Sigma-Aldrich, we collected quercetin (Q-4951) (St. Louis, MO, USA). After being dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), the product was used to make a stock solution with a concentration of 1 mg/mL. The MIC was verified and very slightly modified from previous study [37 (link)]. A two-fold serial dilution approach using TSB was used to establish the minimum inhibitory concentration (MIC) of quercetin against V. parahaemolyticus. A total of 100 µL of quercetin serially diluted with TSB and 100 µL of bacterial suspension (10⁵ log CFU/mL) were combined in 96-well plates (Corning Incorporated, Corning, Inc., Corning, NY, USA). Each well had a total amount of 200 µL. A microplate reader (Spectra Max 190, Sunnyvale, CA, USA) was used to measure absorbance (600 nm) while the plates were kept in a 30 °C incubator for 24 h. After an overnight incubation at 30 °C, aliquots (100 µL) taken from the wells that had no discernible growth were plated on Vibrio CHROMagar (CHROMagar, Paris, France) plates and the number of colonies counted. Triplicates of this experiment were performed.

In the subsequent experiments, Ang II at 600 nM was used to induce cell hypertrophy. H9c2 cells were randomly allocated into four different groups: Control, Ang II, Ang II + quercetin (331 μM) [36 (link)], and Ang II + rutin (50 µM) [38 (link)]. The cells were treated with Ang II alone or in combination with either quercetin or rutin for 24 h. The final DMSO concentration used as a vehicle for quercetin and rutin was less than 0.1% [19 (link)]. The concentration of the flavonoids was selected according to the effective concentration reported to reduce cell hypertrophy and cardiac injury in H9c2 cells [36 (link),38 (link)]. Both quercetin (Q4951) and rutin (R5143) were commercially obtained from Sigma-Aldrich (St. Louis, MO, USA).