Celltitre glo assay kit

Cellular viability was measured by bioluminescent detection of ATP (CellTitre-Glo assay kit (Promega)). Cells were plated at 5 × 10³ cells per well in 96-well, white-walled plates, allowed to adhere and treated with ETYA (10 µM) or EtOH (vehicle control) to a final volume of 100 µl for 96 h. Each experiment was performed in triplicate in triplicate wells. For scratch assay, cells were seeded at 1 × 10⁶ cells per well in a 6-well plate, allowed to adhere for 48 h to a confluence of about 80% and then wounded by scratching with p200 sterile pipette tip. The debris were removed, and cells washed to make ensure the edges were smoothed with the same dimensions for experimental and control cells. Cells were incubated, and cell migration was assessed by monolayer gap closure after 48 h.

MCF-10a and MDA-MB-231cells were transfected with the constructs or miRs indicated in the appropriate Figure legend using Lipofectamine. After 24 h, cells were trypsinized, and 1 x 10⁵ cells were seeded on transwell chambers precoated with 20 μg Matrigel for MDA-MB-231 cells and 10 μg Matrigel for MCF-10a cells. FCS (10%) in the lower chamber served as chemoattractant. After an additional 24 h, non-invading cells were removed with cotton swabs, and invading cells were trypsinized and counted using the Cell-Titre-Glo assay kit (Promega, USA) as described previously [39 (link)]. Remaining cells were plated into a 6-well plate for protein- and RNA-isolation after 24 h. The same method was applied to analyze MDA-MB-231 cells stably expressing shRNAs and MCF-10a cells stably expressing GNA13.

A549 cells were seeded at 1 × 10⁴ cells/well in a 96 well plate before being treated with GS-9 (250 μM) in the presence of increasing concentrations of Vitamin E for 16 h before the CellTitre-Glo assay kit (Promega, Madison, WI, USA) was used to determine viability of treated cells. All values were corrected to a blank control and absorbance of the vehicle control was designated as 100% viability.