Computational protocol: TCF4-Targeting miR-124 is Differentially Expressed amongst Dendritic Cell Subsets

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Protocol publication

[…] Single cell suspensions from the harvest of mouse organ tissues or cultures thereof were incubated with 2.4G2 (Fc blocker) hybridoma supernatant and washed with FACS buffer (2% FCS, 2 mM EDTA, 0.1% sodium azide). Then, each sample was incubated with the appropriate mixture of fluorochrome-conjugated monoclonal antibodies and live/dead staining dye for 30 min at 4℃ and washed twice with FACS buffer. The samples were then analyzed with FACSVerse flow cytometer (BD Biosciences) or sorting with FACSAria II cell sorter (BD Biosciences). As for sorting, the isolated cells with 90% or higher purity were utilized for subsequent experiments. Gating criteria for respective precursor and DC populations were as follows. With the lineage (Lin) markers of CD3, CD19, Ly6G, NK1.1, DX5, Ter119: for pDCs, Lin− CD11c+ B220+ PDCA-1+ SiglecH+; for cDC1, Lin− CD11c+ MHCII+ B220− CD24highCD172α−; for cDC2, Lin− CD11c+ MHCII+ B220− CD24intCD172α+. With the lineage markers of CD3, CD19, Ly6G, NK1.1, DX5, Ter119, MHCII, B220: for MDP, Lin− CD11c− CD115+ CD135+ CD117high; for CDP, Lin− CD11c− CD115+ CD135+ CD117int. Flow cytometric data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). [...] Experiments with multiplicated samples were presented as mean±SEM from at least three independent experiments. Statistical comparisons between different groups were analyzed using unpaired Student's t-test using SigmaPlot (Systat Software, San Jose, CA). Statistical significance is denoted by the p values equal or below 0.05 (*), 0.01 (**), and 0.001 (***). Data were plotted for graphs with SigmaPlot. […]

Pipeline specifications

Software tools FlowJo, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus