Polystyrene facs tubes

Viability of RPTEC/TERT1 cells was determined by propidium iodide (PI) (Molecular Probes, Oregon, USA) staining. In brief, following trypsinization and centrifugation, cell pellets were re-suspended in culture medium and incubated for 15 min at 37 °C to restore membrane integrity. The cell suspensions were then washed and re-suspended in FACS buffer containing 2% FCS (Gibco) and 0.05% NaN₂ (Sigma) in PBS (Sigma). Cells were transferred as 100-μl aliquots into 5-ml polystyrene FACS tubes (Sarstedt). Finally, PI solution was added to final concentration of 1 μg/ml and the samples were analysed on an Accuri-C6 flow cytometer (Becton Dickinson, USA) using CFlow software.

THP-1 macrophages were stained with eBioscience Fixable Viability Dye (FVD) eFluor 660 (Thermo Fisher), detached with Accutase (Sigma-Aldrich), and transferred to polystyrene FACS tubes (Sarstedt). Single-cell suspensions were washed with MACS buffer (0.5% bovine serum albumin, 2mM EDTA in PBS, ph7.2) at 290 x g for 5 minutes at 4°C, and in most cases, fixed in 4% PFA solution overnight at 4°C. The following day, samples were washed with and resuspended in MACS buffer. Data were acquired in LSRII or Fortessa flow cytometers (BD Biosciences) and analyzed in FlowJo 10 (TreeStar), gating on single-cell events.