Anti cd45 apc vio770

Purity and phenotype of nDCs after immunomagnetic isolation were determined by flow cytometry with a FACSVerse® (BD biosciences, San Jose, CA) or MACS Quant® (Miltenyi Biotec). For this purpose, the following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright-FITC, anti-BDCA2-PE, anti-CD123-APC, anti-CD20-PE-Vio770, anti-CD45-APC-Vio770, anti-CD14-Viogreen, anti-FcεRI-BioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-VioBlue, anti-HLA-ABC-APC, anti-HLA-DR/DP/DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC, and anti-CD86-APC (all Miltenyi Biotec). Details are depicted in Supplementary Table 2. The purity of the nDC product was defined as the percentage of nDCs (sum of CD123⁺BDCA2⁺ pDC plus CD1c⁺CD20^- cDC2) of all viable cells in the nDC product. After 6 h of protamine/mRNA stimulation, cytokine production of nDCs was measured in the supernatant by cytometric bead array according to the manufacturer’s instruction (Miltenyi Biotec).

The antibodies used for blast cell identification by Flow Cytometry (FACSCanto II, Becton Dickinson, Rutherford, NJ, USA) were: anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC all from Miltenyi Biotech (Bergsch gradbach, Germany). For Western blot analysis, anti-β-catenin, anti- Ser675-phospho-β-catenin, anti-Ser33/37/Thr41-phospho-β-catenin, anti-non-phopho-β-catenin, anti-GSK-3β, anti-Ser9-phospho-GSK-3β, anti-GSK-3α, anti-Ser21-phospho-GSK-3α anti-Histone H3 antibodies and Alexa 488-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA); anti-GAPDH, and HRP-conjugated secondary antibodies against mouse or rabbit were from Sigma-Aldrich (Darmstadt, Germany). Wnt modulators used for proliferation and vitality assays, i.e., Wnt-3a, PNU-74654, Niclosamide, IWP-2, Lithium Chloride (LiCl), and AR-A014418, were all purchased from Sigma-Aldrich. For the analysis of cell death, Propidium iodide (PI) and FITC-conjugated Annexin V were from Miltenyi Biotechnology. CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS, Eden Prairie, MN, USA) was from Promega (Promega, Milano, Italy). Cytarabine (Ara-C) and Idarubicin (Ida) were provided by the Pharmacy Unit of the University Hospital of Verona.

Purity and phenotype of mDCs and pDCs after CliniMACS isolation were determined by flow cytometry with a FACSVerse (BD Biosciences, San Jose, CA, USA) or MACS Quant (Miltenyi Biotec). The following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright FITC, anti-BDCA-2-PE, anti-CD20-PE-Vio770, anti-CD123-APC, anti-CD45-APC-Vio770, anti-CD14-VioGreen, anti-FcεRI-VioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-BioBlue, anti-HLA-ABC-APC, anti-HLA-DR,DP,DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC and anti-CD86-APC (all Miltenyi Biotec).

Flow cytometry was performed the same day as collection to determine cell populations in whole blood, peritoneal lavage, abdominal wall, and liver tissue. Tissue was dissociated to obtain a single-cell suspension by Liberase TM research-grade solution (1:100, Roche, Basel, Switzerland) in RPMI medium and filtration through a 0.3 μm membrane filter (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Tissue cell suspension and peritoneal lavage were centrifuged at 380 RCF for 5 min at RT to obtain a cell pellet, which was then resuspended in FACS buffer to a final concentration of 10⁶ cells/mL for further analysis. Cells in the single cell suspensions were blocked with 10% mouse serum for 10 min at 4 °C to prevent unspecific binding. Cell suspension and full blood were incubated for 15 min at 4° with the combination of fluorochrome-conjugated antibodies: anti-CD45-APC-Vio770 (1:50, clone REA737), anti-Ly-6G-FITC (1:50, clone REA526) (both Miltenyi Biotec, Bergisch Gladbach, Germany), and propidium iodide solution (1 μg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometry was performed using MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany), compensation was applied and cell populations with corresponding data were analyzed with FlowJo (version 10.5.3, Becton, Dickinson and Company, Ashland, OR, USA).

The antibodies used for FACS analysis were: mouse IgG2b-FITC, goat IgG-PE, anti-Jagged1-FITC, anti-Dll3-PE (all from R&D System, Minneapolis, MN), mouse IgG2a-PE, mouse IgG1κ-PE, mouse IgG1-Alexa Fluor 488,anti-Notch1-PE, anti-Notch2-PE, anti-Notch3-PE, anti-Notch4-PE, anti-Dll1-PE, anti-Dll4, anti-Bax-Alexa Fluor 488 (all from Biolegend, San Diego, CA) and rabbit anti-Bcl-2-FITC (DAKO). For leukemia cell identification we used anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC (all from MiltenyiBiotec, Germany). The antibodies employed for western blot analysis anti-Notch2, anti-Notch4 were from Santa Cruz (Biotechnology, Dallas, TX), anti-GAPDH and HRP conjugated secondary antibodies against mouse, rabbit or goat were from Sigma Aldrich. All the other antibodies used for Western blot were from Cell Signalling. Neutralizing antibodies,all used at a final concentration of 5 μg/ml, were: anti-Notch1, anti-Notch3,anti-Jagged1, anti-Jagged2, anti-Dll1 and anti-Dll4 (R&D Systems); anti-Notch-4 (Santa Cruz Biotechnology); anti-Dll3 (CST, Boston, MA). Recombinant human Jagged-1 and Jagged-2 were from R&D System. GSI-IX (DAPT) was purchased from Stemgent (Cambridge, MA) GSI-XII and SAHM1 were from Merck Millipore (Darmstadt, Germany). Cytarabine (Ara-C), Etoposide (Eto) and Idarubicin (Ida) were provided by Pharmacy Unit of the University Hospital of Verona.