Computational protocol: Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development

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[…] Fluorochrome-conjugated antibodies specific for CD11c (N418), CD11b (M1/70), CD115 (AFS98) and streptavidin-APC-Cy7 were obtained from eBiosciences or Bioloegend (San Gabriel, CA, USA). Fluorochrome-conjugated antibodies specific for CD8α (53–6.7), B220 (RA3-6B2), MHC-II (AF6-120.1), CD3ε (145-2C11), c-kit (2B8), CD16/32 (Clone 93), Gr-1 (RB6-8C5), Sca1 (E13-161.7), CD34 (RAM34), IL-7Rγ (A7R34), Flt3 (A2F10), CD150 (TC15-12F12.2), Ly6C (HK1.4), Ly6G (1A8), streptavidin-PE-Cy7, streptavidin-PE and streptavidin-FITC were obtained from Biolegend. Goat-anti rat-PE-Texas Red was obtained from Invitrogen (Eugene, OR, USA). Isotype control antibodies including Rat IgG2a-FITC (R35-95), Rat IgG2b-PE (RTK4530), Rat IgG2b-PE-Cy7 (eB149/10H5), Mouse IgG2a-biotin (eBM2a) and Hamster IgG-APC (eBio299Arm) were obtained from eBiosciences.Cells were stained as previously described [, ]. Briefly, 1 x 105–1 x 106 cells were incubated with purified CD16/32 antibody (Clone 93; eBiosciences) for 15 min to block surface Fc receptors. Cells were then washed with DMEM/1%FCS/0.1%NaN3 and stained with primary antibodies for 20 min on ice. Any secondary reagents were added to the stained cells after a washing step, and further incubated for 20 min on ice. Cells were washed twice and resuspended in DMEM/1%FCS/0.1%NaN3 for flow cytometric analysis using a LSRII flow cytometer (Becton Dickinson). Cells were stained with PI (1μg/ml) for live cell discrimination. Gates were set to delineate cell subsets using isotype control antibodies and ‘fluorescence minus one’ controls. Cell subset analysis was performed using BD FACSDiva Software (Becton Dickinson) and FlowJo Software (Tristar; Phoenix, Arizona, USA). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus
Diseases Carcinoma, Renal Cell, Splenic Diseases