Attune cytometric software v2

MCF-7 cells seeded in Petri dishes were treated during 72 hours with indicated compounds. After trypsinization and permeabilisation/fixation with 70% ethanol, cells were treated with DNase-free RNase (Invitrogen) and DNA was stained with propidium iodine (Invitrogen) at 10 μg/mL for 15 minutes. After a 2x dilution in PBS, cells were analyzed by flow cytometry. Cytometric analyses were carried out with an Attune acoustic focusing cytometer and data were analyzed using Attune cytometric software V2.1 (Applied Biosystems).

Cells were seeded in 6-well plates (2.5 10⁵ cells/well) and allowed to grow for 24 hours in serum-complete condition. Then, cells were treated or not (control) for a 24-hour period (serum-complete condition). After monolayer trypsinization, cells were labeled with annexin V-FITC and propidium iodine (Dead Cell Apoptosis, Invitrogen) according to manufacturer's instructions. Cytometric analyses were carried out with an Attune acoustic focusing cytometer and data were analyzed using Attune cytometric software V2.1 (Applied Biosystems). Statistical analysis was performed by ANOVA followed by post hoc Tukey test (for p values <0.05) using Fizz software.

Cell surface staining was performed using monoclonal antibodies fluorescently labeled with fluorescein isothiocyanate (FITC), allophycocyanin (APC) or phycoerythrin (PE). Anti-CD3 and anti-CD14 antibodies (ImmunoTools GmbH, Friesoythe, Germany) were used to stain T cells and monocytes, respectively. Anti-CD86 antibody (ImmunoTools), and anti-HLA-DR (Immunostep; Salamanca, Spain) were used to assess MoDC maturation. Data were acquired with Attune® Acoustic Focusing Cytometer and analyzed using Attune® Cytometric Software v2.1 (Applied Biosystems, Carlsbad, CA, USA. In each case, at least 10,000 events were acquired in the gate of interest. The forward scattered (FSC) and the side scattered (SSC) light were used to evaluate relative cell size and granularity and cell doublets were excluded based on FSC-A/FSC-H. Mean Fluorescence Intensity (MFI) was determined for each marker.