Facs caliber bench top flow cytometer

Cells treated with DMSO or BKM1644 were trypsinized and washed with PBS and resuspended in Annexin-binding buffer (BD Pharmingen, San Diego, CA). Cells were then stained with both Annexin V-phycoerythrin and 7-amino-actinomycin for 15 min at room temperature. The stained samples for apoptosis assay were measured using a fluorescence-activated cell sorting (FACS) caliber bench-top flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

Apoptosis was analyzed in all four cell lines. Cells were treated with honokiol, paclitaxel, or their combination as indicated in the figure legends, trypsinized, and washed in cold 1× PBS. The cells were resuspended in 1× Annexin binding buffer (BD PharMingen), and then stained with Annexin V-phycoerythrin (Annexin V-PE; BD PharMingen) and 7-AAD (BD PharMingen) for 15 min at room temperature. The stained samples were measured using a fluorescence-activated cell sorting (FACS) caliber bench-top flow cytometer (Becton Dickinson, Franklin Lakes, NJ). FlowJo software (Tree Star, Ashland, OR) was used for apoptosis analysis. The experiments were repeated 3 times independently.

A549^pBabe and A549^LKB1 cells were treated with either diluted DMSO, selumetinib alone, phenformin alone, or their combination as indicated, and cells were collected after 48 hours by trypsinization, washed with cold × 1 phosphate-buffered saline (PBS), and stained with Annexin V-phycoerythrin (PE) and 7-AAD (BD PharMingen) for 15 minutes at room temperature. The apoptotic population of the samples was then measured using a fluorescence-activated cell sorting (FACS) caliber bench-top flow cytometer (Becton Dickinson). FlowJo software (Tree Star) was used for apoptosis analysis.