Reafinity

Reference concentrations of the CD4⁺ T lymphocytes in the WB samples were determined with an optical flow cytometer (MACSQuant Analyzer 10, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). Therefore, 20 µL WB was stained with 1 µL each of anti-CD4 antibody conjugated with FITC fluorophore (REAfinity, Miltenyi Biotec B.V. & Co. KG), anti-CD3 antibody conjugated with APC-Vio 770 fluorophore (REAfinity, Miltenyi Biotec B.V. & Co. KG), and anti-CD45 antibody conjugated with VioBlue fluorophore (REAfinity, Miltenyi Biotec B.V. & Co. KG). After 30 min incubation, the sample was diluted at a ratio of 1:30 with MACSQuant Running Buffer (Miltenyi Biotec B.V. & Co. KG) and immediately measured.

Cells (1 × 10⁴ cells/plate) were seeded in a 35 mm glass bottom cell culture dish in 500 µL complete medium and incubated at 37 °C. Three days later, cells were induced for stress with IL-1β for HTB161 and with IL-1β+TNFα for HTB75. Treatments were given after 48 h of induction, for 16 h at sub-lethal concentrations. For fixation and permeabilization of cells, Transcription Factor Staining Buffer Set (130-122-981; Miltenyi Biotech, Bergisch Gladbach, Germany) was used, and cells were incubated for 30 min at 4–8 °C. For staining, β-Catenin monoclonal antibody (130-121-990-PE; REAfinity™ Miltenyi Biotech, Germany) was used, and cells were incubated for 40 min at 4–8 °C. Hoechst (EasyProbes™ 33342; ABP Bioscience, Beltsville, MD, USA) was used for nuclear staining. Cell microscopy and image acquisition was carried out using a Leica SP8 laser scanning microscope (Leica, Wetzlar, Germany), equipped with 405 and 552 nm solid state lasers, HC PL APO CS 63×/1.2 water immersion objectives (Leica, Wetzlar, Germany) and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). Hoechst, 5(6)-Carboxyfluorescein and PE (red)emission signals were detected with PMT and HyD (hybrid) detectors in ranges of 415–490 nm, and 565–660 nm, respectively.