Live dead fixable violet cell stain

Exponentially dividing MDA MB 231 cells were trypsinized and re-suspended into a 1 × 10⁶ cell/100 μl PBS. MitoTracker (0.125 μl/ml) was added to cell suspension for 30 mins at 37°C before centrifugation and re-suspension in 100 μl PBS and the addition of 10 μl ESA-FITC (DAKO, BerEP4), 10 μl CD24-PE (Pharmingen) for 10 mins at 4°C before centrifugation and re-suspension in PBS for FACS analysis (BD, LSR Fortessa. All tubes were incubated with 1 μl/ml live/dead fixable violet cell stain (Molecular Probes) to exclude dead cells from FACS analysis and sorting.

Transfected THP-1 cells were recollected at different times and stained with LIVE/DEAD Fixable Violet Cell Stain (Molecular Probes Life Technologies) for 30 min. Cells were washed twice and fixed with 2% PFA. Cells were examined in a Gallios flow cytometer (Beckman Coulter). Data was analyzed with FlowJo software (V.10.2) to determine the percentage of GFP-positive cells and its correlation with death in triplicate samples.

Freshly thawed PBMCs were labeled with CellTrace Violet (Invitrogen) following the manufacturer's instructions and cultured for 6 days at 37°C and 5% CO₂ in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 20 units/ml of IL-2 (Sigma), in the presence or absence of 10 μg/ml of peptide. The PBMCs were then re-stimulated with peptide in the presence of monensin (Golgi Stop, BD Biosciences, San Jose, CA) for 12 hours. Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences) at room temperature for 15 minutes in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry analysis. Intracellular staining was performed in Perm/Wash (BD Biosciences), and the following mAbs were used, CD3 PerCP-Cy5.5 (clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), and IFNγ APC (clone B27), all from BD Biosciences. Live/dead Fixable Violet Cell Stain was from Life Technologies (Eugene, OR). All data was acquired on a BD LSRFortessa X-20 instrument (BD Biosciences) and analyzed using FlowJo Version 9.9.4 software (TreeStar, Ashland, OR).