Biotin conjugated mouse lineage panel

To analyze neutrophils, single cell suspensions of bone marrow or peripheral blood or spleen were stained with CD11b PerCP-Cyanine5.5 (eBioscience 45-0112-82) and Ly-6G-APC (eBioscience 17-5931-82). To measure neutrophil infiltration in the lung, lung tissues were cut into very small fragments and digested by collagenase and DNase I for 20 minutes at 37°C. Single cell suspensions were then stained with CD45-FITC (BD pharmingen, 553080), Ly-6G-APC and CD11b PerCP-Cy5.5. To detect the myeloid progenitor cells, bone marrow cells were pre-stained with biotin-conjugated mouse lineage panel (BD pharmingen, 559971), and then stained with streptavidin-V450 (BD horizon, 560797), Sca-1-PE-Cy7 (BD pharmingen, 558162), c-Kit-PE (BD pharmingen, 553355), CD34-FITC (BD pharmingen, 560238) and CD16/32-APC (eBioscience, 17-0161-82). Flow cytometry was conducted on a FACS Aria (BD Biosciences).

Primary BM isolates were depleted of Lin^- cells by negative immunomagnetic selection using the EasySep Biotin Selection kit (Stem Cell Technologies, Cambridge, MA, USA #18556) according to the manufacturer’s instructions. Biotin antibodies used for Lin positive selection were included in the Biotin-conjugated Mouse Lineage Panel (#559971 BD Pharmingen). Recovered cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% FBS and incubated with conjugated primary antibodies to MSC-associated cell surface markers for 30 min at room temperature. Unbound antibodies were removed by washing. Cells were detected using an LSRII FACSdiva flow cytometer (BD Biosciences). The antibodies used were CD54 (FITC-conjugated rat IgG2b), CD105 (PE-conjugated rat IgG2a, k), CD73 (PE-conjugated rat IgG1) (all from eBioscience, Thermofisher, Waltham, MA, USA), and CD106 (Alexa 488-conjugated rat IgG2a, k) (Biolegend, San Diego, CA, USA).