Computational protocol: Altered pattern of monocyte differentiation and monocyte-derived TGF-β1 in severe asthma

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Protocol publication

[…] For simultaneously staining PM-2K+ cells and fibrocytes, fluorochrome-conjugated monoclonal antibodies against the following antigens were used: CD3, CD14, CD19, CD45RO, collagen I and a specific marker of macrophages, PM-2K. Ficoll-isolated PBMCs (Ficoll-Paque Plus, GE Healthcare, Biosciences, Piscataway, NJ) were sequentially stained with human Fc receptor binding inhibitor (eBioscience), purified anti-macrophage Abs (PM-2K, Serotec) and followed by anti-mouse IgG-FITC. After washing, the cells were then fixed and stained intracellularly with rabbit IgG anti-collagen I (Rockland) and anti-rabbit IgG-Qdot655 (Invitrogen) using intracellular staining kit (eBioscience). After washing, the cells were then stained with other fluorochrome-labeled monoclonal antibodies against surface markers in PBS containing 0.5% fetal bovine serum, including CD3-Pacific blue (UCHT1, BD), CD19-Pacific blue (HIB19, eBioscience), CD14-PE/Cy7 (61D3, eBioscience), and CD45RO-PE (UCHL1, BD) and appropriate isotype controls. For analysis of PM-2K+ subsets, PBMCs were stained with PM-2K antibody, followed by anti-mouse IgG-FITC. After washing, cells were then stained with fluorochrome-conjugated monoclonal antibodies against surface markers, including CD3, CD19, CD14, CD86 and CCR7.FMO controls specific for PM-2K, collagen I, CD86, or CCR7 markers for each sample were used to facilitating recognition of boundaries between positive and negative subsets. For example, to determine the positive boundary for collagen I expression in CD3−CD19−CD45+ cells (Fig. ), a FMO control was prepared in which cells were stained with all fluorochrome-conjugated monoclonal antibodies except the one that recognizes collagen I. The samples were run on a LSRII flow cytometer (BD Biosciences, San Jose, CA) with data acquisition on 106 live cells (gated by forward and side scatter properties) and analyzed using FlowJo software (Tree Star, Inc, Ashland, OR). Moreover, the detailed technical protocols regarding the blocking step after the anti-mouse IgG-FITC staining and the fixation effect on staining pattern are provided in Supporting Information with pertinent data shown in Supplementary Fig. , Figs.  and , respectively. [...] Statistical analysis was performed using IBM SPSS Statistics (Version 19, IBM Company, Armonk, NY, USA) and GraphPad Prism (Version 5, GraphPad Prism Software, Los Angeles, CA, USA). Mann-Whitney U test was used to determine the difference between normal subjects and patients. Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test was used to determine the difference between subgroups of patients. Spearman correlation test was used to determine the correlation between the indicated subsets and ACT scores as well as other clinical parameters (severity and lung function tests). P value < 0.05 was considered significant. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry