Cytofix citoperm

Cells were fixed and permeabilized with Cytofix/Citoperm™ (BD Bioscience, Erembodegem, Belgium) and stained with the following primary antibodies: anti-β catenin, anti-LEF1, anti-TCF4 and anti-WNT5a (Santa Cruz Biotecnology). After washing cells were incubated with secondary antibodies. After washing cells were then analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Median Fluorescence Intensity (MFI) was evaluated on a linear scale. Cell death and apoptosis were analyzed by annexin-V FITC/propidium iodide (PI) double staining method. Cells were harvested by trypsinization, resuspended in the staining buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), stained with FITC-labeled annexin V and PI for 15 min at RT in the dark and then kept on ice until be analyzed. Data from 5×10⁴ cells were acquired from each sample and analyzed using FlowJo software.

After separation from the whole blood in EDTA, 100 μl of viable PBMC (peripheral blood mononuclear cell) was stained with different combinations of monoclonal antibodies. To characterize the phenotype of T and B cell subsets, extracellular labeling was performed with anti-CD8_FITC, anti-CD161_PE, anti-CD3_ECD, anti-CD4_PC5.5, anti-CD16_FITC, anti-CD56_PE, anti-IgD_FITC, anti-CD27_PC5.5, and anti-CD19_ECD (Beckman Coulter, Miami, FL). Living cells were gated within the side/forward scatter (SSC/FSC) lymphocyte gate. For intracellular staining, cells were permeabilized with Cytofix/Citoperm (BD Biosciences). Finally, the cells were stained with anti-IL-17A_FITC (MiltenyiBiotec), washed, and analyzed. All measurements were made with a CyAN ADP flow cytometer (Beckman Coulter, Miami, FL, USA) with the same instrument setting. At least 105 lymphocytes were acquired and analyzed using FlowJo (Tree Star) software. Leukocyte count and differential were determined with a routine hematology analyzer. The absolute counts of total lymphocytes were calculated by multiplying the relative size of the T and B cells and the absolute lymphocyte count.

Cells being studied were cultured in 12-mm coverslips with or without NeoSTX (1 µM) for 24 h in DMEM or supplemented RPMI media. They were then washed with 1X PBS and fixed with absolute ethanol for 10 min at room temperature. After that, they were washed with PBS 1X and fixed with Cytofix/Citoperm (BD, Biosciences, 554722) for 20 min at 4 °C. They were, subsequently, washed again with Perm/Wash Buffer 1X (Biosciences, 554723) and the primary anti-mouse anti-Nav 1.6 1:100 antibody (Abcam, catalog ab65166) or the anti-NeoSTX antibody (Membrane Biochemistry Laboratory, Universidad de Chile) diluted in Perm/Wash Buffer 1X for 20 min at 4 °C. After this time, the secondary polyclonal anti-rabbit FITC 1:100 anti-body (Dakocymation F0205) was added, washed again with Perm/Wash Buffer 1X and mounted with DAPI 1:200 (BD Pharmingen, 564907) for 15 min. The analysis was performed in a Zeiss brand confocal microscope, model 710, and Zen software was used for data analysis.