Sodium azide 1 15n

HeLa cells were obtained from the laboratory of Lars Jansen at Instituto Gulbenkian de Ciência (IGC) and maintained in Dulbecco's modified Eagle medium high glucose with L-glutamine and sodium pyruvate (Biowest) supplemented with 10% fetal heat-inactivated (Biowest) and 1% penicillin–streptomycin (Biowest)—Dulbecco's modified Eagle medium complete medium. Cells were maintained in humidified atmosphere, 5% CO₂ at 37 °C. Cells were counted in a haemocytometer (Neubauer Cell with Double Grid, Fisher Scientific) using 1% trypan blue solution (Sigma-Aldrich) and plated at 10⁵ and 6 × 10⁴ cells per well in 12- and 24-well plates (CORNING), respectively.
All incubations used a concentration of 6.50 nM of DT and PEGyDT. Different time points (6–72 h) were prepared for FACS analysis. The medium and cells were collected and cells were centrifuged for 4 min, 3,250g, at 4 °C. Cells were resuspended in 150 μl FACS buffer (2% FBS, 0.02% sodium azide-1-¹⁵N (Sigma-Aldrich) in PBS 1x) and 25 μl propidium iodide (Sigma-Aldrich) were added.
Data were acquired in a Becton Dickinson Flow Cytometer FACSCalibur. Data were acquired using CellQuest software (Becton Dickinson) and analysed in FlowJo.