Nuclear factor fixation and permeabilization buffer set

Cells were cultured in 6-well trays and treated with drugs as described above. After 24 and 48 hours of treatment, cells were fixed and permeabilised using Nuclear Factor Fixation and Permeabilization Buffer Set (Biolegend, CA, USA) according to the manufacturer's instructions. 2×10⁵ cells were labelled either with anti-p53-PE or mouse IgG2b-PE (1 μg/mL; Biolegend) and the cells were sorted and the data were acquired on an LSRII, and the data were analysed using FlowJo software.

(1) Cells were detected by size and granularity using FSC/SSC, and cell debris was gated out. The cell size was evaluated by cytometric light scattering of PI-negative stained cells [67 ]. To discriminate the live and dead cells, two-parameter histogram (DotPlot or Cytogram) was used (FL4LOG vs. FSLOG). (2) The samples were prepared using Nuclear Factor Fixation and Permeabilization Buffer Set (BioLegend, USA). Briefly, after fixation and permeabilization cells were stained with FITC-Ki67 conjugate (Dako) (10 μl/10⁶ cells) and DAPI (1 μg/ml). FITC Mouse IgG1 (BD Pharmingen) served as a negative control [68 (link), 69 (link)]. (3) After trypsinization and washing with PBS, MESCs were concentrated to 1x10⁶ cells per ml in FACS buffer, and then stained with CD146 antibody (Beckman Coulter, USA) conjugated with phycoerythrin in accordance with manufacturer’s recommendations at least for 40 min at +4°C in the dark. Phycoerythrin conjugated Mouse IgG1 used as isotype control. The percentage of expressed cell surface antigens was calculated for 10, 000 gated-cell events [62 (link), 70 (link), 71 (link)]. In all indicated cases, flow cytometry was performed using the CytoFLEX (Backman Coulter, CA, USA), and the obtained data were analyzed using CytExpert software version 2.0.