Mouse igg1 alexa fluor 488

Collagen I from human skin, Pronase, DNase I, SBTI, accutase, and IgG from human serum were purchased from Sigma-Aldrich; PneumaCult™-EX PLUS, PneumaCult™-ALI were from STEMCELL Technologies; EASYstrainer Cell Sieves (100 and 40 μm), transwell inserts and 96 well plates with cell-repellent surface were from Greiner Bio-One; High Pure RNA Isolation kit, Transcriptor First Strand cDNA synthesis kit, SYBR Green PCR Master Mix were from Roche Diagnostics.
Antibodies for flow cytometry staining were ACE2-AlexaFluor 647 (clone E-11), AXL-AlexaFluor647 (clone B-2), acetylated α-Tubulin-AlexaFluor 488 (clone 6-11B-1), MUC5AC-AlexaFluor 488 (clone 45M1), CC10-AlexaFluor 488 (clone E-11), Cytokeratin 5-AlexaFluor 488 (clone RCK103) were purchased from Santa Cruz Biotechnology; Mouse IgG1-AlexaFluor 488, Mouse IgG1-AlexaFluor 647 were from BioLegend, Mouse IgG2b-FITC was from DAKO; and Live/Dead™ Fixable Blue Dead Cell Stain was from Invitrogen.

The antibodies used for flow cytometry were: mouse IgG2b-FITC, goat IgG-PE, anti-Jagged1-FITC, anti-Dll3-PE (all from R&D System, Minneapolis, MN, USA), 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, (all from Biolegend, San Diego, CA, USA) (DAKO). For blast cell identification, we used anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP, and anti-CD117-APC (Miltenyi Biotec, Germany).

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.