Facs cyan flow cytometer

We evaluated cell surface marker expressions on cultured human macrophages using monoclonal antibodies (mAbs) to CD80, CD86, CD11b, CD68, CD14, CD16, HLA-ABC, HLA-DR, CD1a, CD1b, CD1c, CD1d, the TCRβ chain, TCRγδ, CD3ε (epsilon chain), CCR4, CCR7, CXCR1, and TNF. Likewise, to evaluate mouse myeloid cells, we used mAbs to CD11b, CD3ε, TCRβ, TNFR1, and TNFR2. All the mAbs were provided by BioLegend (San Diego, CA, USA). The cells were stained for 30 min, at 4°C in the dark. Then, the cells were fixed by 2% p-formaldehyde in phosphate-buffered saline (PBS: 10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.2). The cells used for Fluorescence Minus One (FMO) condition were stained and acquired in parallel to identify background levels of staining, dead cells were omitted by use of Zombie Aqua^TM (BioLegend) viability kit.
The data were collected by means of a FACS Aria II (BD Biosciences, San Jose, CA, USA) or FACS CyAn flow cytometer (Beckman Coulter, Inc. Brea, CA, USA) and analyzed by FlowJo v10.2 (FlowJo LLC, Inc, Ashland, OR, USA). In each case, 50,000 events were acquired per sample. A list of the antibodies clones used can be found in Table S1.

The frequency of immunological cellular subpopulations in pleural cells was analyzed by flow cytometry. Briefly, cells were stained for 30 min at 4 °C with following fluorochrome-conjugated mAb: CD11b (Clone M1/70) and TNFR2 (Clone TR75-89) (BioLegend, San Diego, CA, USA). Cells were incubated with antibodies and then washed with PBA (phosphate buffered saline containing 0.1% Sodium Azide and 1% Albumin bovine). We used a FACs CyAn flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and analyzed the data with the FlowJo (Tree Star, Ashland, OR, USA) software. We acquired 100,000 events per sample.