Doxorubicin hydrochloride powder

Alamar blue (AB) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays were performed in 96 well plates and a total number of 1 × 10 5 cells were used to seed three plates (4 × 10 3 cells mL -1 ). Doxorubicin hydrochloride® powder (Sigma Life Sciences, Ireland) was diluted in 1 mL sterile water to the required concentration. After 24 h incubation, plates were washed with phosphate buffered saline solution (PBS) and doxorubicin was added in a range from 0 µM (as a control) to 50 µM.
A solution of 1.5 mL of AB (10× ready to use solution) and 3 mL of MTT stock solution (2.5 mg mL -1 , 25 mg MTT/10 mL PBS) in 30 mL of fresh medium were prepared. AB and MTT assays were both measured with a Cytotox SpectraMax®M3 plate reader using Soft Max® Pro6.2.2 as software and data was treated using SigmaPlot 10.0. After 24 h incubation in DOX, plates were washed with PBS and 100 µL of AB/MTT solution were added to each well. Plates were then incubated for 3 hours and AB fluorescence was measured before in the plate reader using 540 nm excitation and 595 nm emission. The medium was then removed, the plates were washed with PBS and 100 µL of DMSO (dimethyl sulfoxide) were added in each well. MTT absorbance was read at 570 nm. All cytotoxicity assays were made in triplicate and repeated three times.

A549 human lung adenocarcinoma cells with the alveolar type II phenotype were obtained from ATCC (Manassas, VA, USA). Doxorubicin hydrochloride ® powder (Sigma Life Sciences, Ireland) was diluted in1mL sterile water to the required concentration (17.25 mM).
Alamar blue (AB) (10X ready to use solution) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Aldrich, Ireland.
For cytotoxicity assays, an AB/MTT solution, 1.5mL of AB and 3 mL of MTT stock solution (2.5mg/mL, 25mg MTT/10mL PBS) in 30mL of fresh medium was prepared prior to the tests.

A549 human lung adenocarcinoma cells with the alveolar type II phenotype were obtained from ATCC (Manassas, VA, USA). Doxorubicin hydrochloride ® powder (Sigma Life Sciences, Ireland) was diluted in1mL sterile water to the required concentration (17.25 mM).
Alamar blue (AB) (10X ready to use solution) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Aldrich, Ireland.
For cytotoxicity assays, an AB/MTT solution, 1.5mL of AB and 3 mL of MTT stock solution (2.5mg/mL, 25mg MTT/10mL PBS) in 30mL of fresh medium was prepared prior to the tests.