Infinicyt software

For each AML case, we requested that the participating center upload the anonymized flow cytometry standard (FCS) files (raw data from the EuroFlow AML panel) to the secured EuroFlow server. In addition, the participating laboratories performed a basic analysis on merged FCS files (removal of debris/doublets and identification of lymphocytes) using only the backbone markers (allowing a uniform analysis across tubes) (Infinicyt software, Cytognos, Salamanca, Spain). The resulting analyzed (CYT) file was also uploaded to the secured server. In addition, a spreadsheet with patient annotations (e.g., age, gender, WHO classification and flow cytometer used) was requested. Finally, each CYT file was checked by an independent reviewer to make sure all cells were gated correctly.

MSC phenotypes, as defined in the ISCT minimal criteria [11 (link)], were tested by flow cytometry. MSCs (~10⁶ cells) were harvested, resuspended in PBS, and incubated with conjugated antibodies using the following panel: CD90-FITC, CD14-PE, CD45-PerCP/CD34-FITP, CD73-PE, HLA-DR-PerCP/CD44-FITC, 166-PE, CD19-PerCP, CD105-APC/CD11b-FITC, CD33-PE, 7AAD-PerCP (FITC: fluorescein isothiocyanate, PE: phycoerythrin, PerCP: peridinin chlorophyll protein, APC: allophycocyanin; Becton Dickinson Biosciences). 100,000 cell events per culture were acquired in a FACSCalibur flow cytometer (BD Biosciences) connected to the CellQuest program (BD Biosciences). Fluorescence-based expression of CD markers per event was analysed using Infinicyt software (Cytognos).

Spleen and lymph nodes (popliteal + inguinal) cells were collected and the red blood cells were lysed with buffer containing NH₄Cl. For regulatory T cells analysis, spleen, lymph nodes and CNS-isolated cells were incubated with 0.5 μg of FITC labeled anti-mouse CD4 (clone GK1.5) and 0.25 μg of APC labeled anti-mouse CD25 (clone PC61.5) for 20 min at room temperature. Intracellular Foxp3 transcription factor was detected using Foxp3 PE Staining Set (eBiosciences, San Diego, CA, USA) according to manufacturer's instructions. For DCs analysis, splenocytes were incubated with 0.25 μg of FITC labeled anti-mouse CD11c (clone N418), 0.03 μg of APC labeled anti-mouse MHCII (clone M5/114.15.2) and 0.125 μg of PE labeled anti-mouse CD86 (clone GL1) for 30 min at 4°C. The cells were then washed, resuspended in flow cytometry buffer and fixed in paraformaldehyde 1%. The cells were analyzed by flow cytometry using the FACSCanto II (BD Biosciences, San Jose, CA, USA). Regulatory T cells and DCs analyses were performed by FlowJo software (TreeStar, Ashland, OR, EUA) and Infinicyt software (Cytognos, Salamanca, Spain), respectively. These experiments were performed 18 days after EAE induction except Foxp3+ regulatory T cells (Treg) evaluation in the spleen and lymph nodes that was also assessed at the preclinical phase.

The cells of GS102, GS289, GS79 and GS245 were seeded in 6-well plates at 5x10⁴ cells per well and treated with Scriptaid (0.5 and 1.5μM) or with LBH589 (5nM, 15nM, or 45nM). The cells were harvested after 6 and 24 hours, were washed and incubated for 15 minutes with FACS buffer (PBS/0.25% bovine serum albumin (BSA)/0.05% NaN3/0.5mM Ethylene-di-amine-tetra-acetic acid (EDTA)/2% human serum) containing primary antibodies against integrin αvβ3 (mouse anti-CD51/CD61, 1:50, Abcam) and CAR (rabbit anti-CAR, H-300, 1:50, Santa Cruz). After washing with FACS buffer, the cells were incubated with secondary antibodies Alexa-488 anti-rabbit and PE-anti mouse (Life Technologies). After staining and fixing with BD FACS lysing buffer (BD Biosciences, San Jose, California) a minimum of 3x10⁴ events were acquired on a FACS Canto II (Becton Dickinson, San Jose, California). The analysis of the flow cytometry data was done using Infinicyt software (Cytognos, Salamanca, Spain). Debris and doublets were removed using FSC-H and FSC-A after which expression was plotted for the remaining events.