Computational protocol: Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin

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Protocol publication

[…] The antibodies used were as follows: α-CD19-FITC (Cat 555412, BD Biosciences, Franklin Lakes, NJ, USA), goat α-rabbit–APC (Cat 3846 Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal α-cytidine deaminase (CDA) (SAB 1300717; Sigma, St Louis, MO, USA), rabbit polyclonal α-deoxycytidine kinase (DCK) (ab151966; Abcam, Cambridge, MA) and α-Actin (A1978-200UL, Sigma). Flow cytometry was performed with the BD LSRII (BD Biosciences). Leukemia burden was quantified by harvesting mouse organs and subjecting cell suspensions to flow cytometry with α-human CD19-FITC antibody or no stain. Data analysis was performed using FlowJo software v10.0.5 (Tree Star, Ashland, OR, USA). For dual PI-γ-H2AX staining, cells were fixed and permeabilised with 1% formaldehyde and 0.1% triton-X. Cells were incubated with 2 µl of primary α-Phospo-Histone γ-H2A.X rabbit mAb (Cat 9718, Cell Signaling Technology, Danvers, MA, USA) for 20 min; and with 1 ul of secondary goat α-rabbit–APC for 20 min. PI was added (final concentration 100 µg/m) before flow cytometric analysis. Cell lysates were prepared for immunoblot using Evans buffer and proteins subject to SDS-PAGE using the NuPage 4–12% Bis-Tris Gel (NP0321, Life Technologies, Carlsbad, CA, USA). Membranes were scanned using the ChemiDoc MP Imaging System (Bio-Rad, Philadelphia, PA, USA). [...] Dose–response curves were used to calculate IC50. The area above the dose–response curve (activity area) was determined as described. Bar graphs, dot-plots and statistics were generated using GraphPad Prism (v6.0c) with two-sample t-tests or one-way analysis of variance and Tukey's multiple comparison tests. Synergy was scored using Chalice software (Horizon CombinatoRx Inc., Cambridge, MA, USA), applying the Bliss-independence model. Bliss-excess synergy scores were summed across matrices to rank combination effects. Scatterplots and heatmaps were drawn with gplots and ggplot2 and hierarchical clustering and correlation analysis were performed in R (v3.1.2). […]

Pipeline specifications

Software tools FlowJo, gplots, Ggplot2
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens, Mus musculus
Diseases Leukemia, Leukemia, Biphenotypic, Acute, Precursor Cell Lymphoblastic Leukemia-Lymphoma
Chemicals Cytarabine, Cytidine