7 aminoactinomycin d

S1T, MT-2, and Jurkat cells (2 × 10⁵ cells) were treated with various concentrations of STF-62247. Apoptotic cells were detected by staining with annexin V-FITC (MBL), 7-amino-actinomycin D (Beckman Coulter, Fullerton, CA, USA), and a MEBSTAIN® Apoptosis TUNEL Kit Direct (MBL) and measuring fluorescence intensity with a Cell Analyzer EC800, as previously described [33 (link)]. Percentages of specific apoptotic cells were calculated as follows: % specific apoptotic cells = (annexin V-positive cells − spontaneous annexin V-positive cells) / (100 − spontaneous annexin V-positive cells) × 100.

Flow cytometry analysis was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells were harvested by trypsin at 37°C for 5 minutes, centrifuged at 400*g and resuspended in 1X PBS supplemented with 1% FCS. 4000 cells were incubated with 10 μL anti-SSEA4-PE (BD Biosciences, Pharmingen™, San Diego, USA), 5 μL IgG1-PE (Beckman Coulter Inc., France), 5 μL anti-CD56-PE (Beckman Coulter Inc., France) and 10 μL anti-CD90-PE (Beckman Coulter, Inc., France) for 15 minutes in a 1.5 mL Eppendorf tube at 4°C in dark. Cell were washed with 1 mL PBS, centrifuged at 400*g and resuspended in 200 μL of 1X PBS in a 96- well round bottom plate. After washing and resuspension, each reaction received 5 μL of viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) and plate was incubated for 10 minutes at 4°C. Cell events were acquired with Guava InCyte™ v.2.3 software. Histograms and dot-plots were generated with a minimum of 3000 events with a sample flow rate of 1.8 μL/mL. Positive staining was obtained by comparison with isotype control set as 99% negative.