Brewer's thioglycollate medium (#225710, BD Biosciences) was diluted 4% (w/v) in distilled water, and then autoclaved. One millilitre of the diluted medium per mouse was injected into the intraperitoneal cavity. After 6 days, peritoneal cells were collected. Intracellular staining was performed with the Cytofix/Cytoperm Cell Permeabilization/Fixation Solution (BD Biosciences). Antibodies used for defining M2 macrophages were resistin-like molecule alpha (ab39628), mannose receptor (clone MR5D3) and CD11b (clone M1/70). Working concentrations of antibodies are listed in Supplementary Table 2 . To define MR+ and RELMα+ cells, positive gates for these markers were set to contain <1% of the negative control samples stained with fluorescence-labelled isotype control IgG. Cells were analysed on a FACS Canto II (BD Biosciences).
Cytofix cytoperm cell permeabilization fixation solution
Manufactured by BD
Cytofix/Cytoperm Cell Permeabilization/Fixation Solution is a laboratory reagent used for the permeabilization and fixation of cells. It facilitates the penetration of antibodies and other molecules into the cells for intracellular staining and analysis.
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2 protocols using cytofix cytoperm cell permeabilization fixation solution
1
Isolation and Phenotyping of M2 Macrophages
2
Evaluating Cytokine Expression in Co-cultured Cells
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Cells were co-cultured with the indicated amount of SA for 24, 48, or 72 h. Cells were treated with 2 μM of monensin (Sigma-Aldrich) for the final 6 h. Cells were washed and incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific) for 30 min. Thereafter, anti-CD45-eFluor 450 (clone HI30), anti-CD31-Super Bright 600 (clone WM-59) (Thermo-Fischer Scientific), and anti-CD90-APC (clone 5E10; BD Biosciences) antibody for 30 min at 4°C. Cells were then fixed with Cytofix/Cytoperm Cell Permeabilization/Fixation Solution (BD) for 15 min at 4°C. Cells were then washed and incubated with anti-IL-6-PE (clone MQ2-6A3, BD), anti-IL-8-PE (Clone 8CH, Thermo Fisher) or anti-IFN-β-Fluorescein (Clone MMHB-3, R&D Systems) for 30 min at 4°C. Purity of primary fibroblasts and level of intracellular cytokine expression were analyzed on a flow cytometer (Attune NxT, Thermo Fisher). Flow cytometry data were analyzed with FlowJo (v10.2, BD).
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