Cd15 pe

In COPD patients, cellular pellet obtained from BALF samples was lysed to avoid red blood cells contamination (RBC lysing solution; BioLegend, San Diego, CA). Cells were resuspended in one ml of PBS supplemented with 2% bovine serum albumin (BSA; Roche Diagnostics GmbH, Mannheim, Germany) to quantify the number of total cells in a MACSQuant cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were adjusted to 1x10⁶cells/ml and stained for 15 min at room temperature in dark with viability dye (Zombie NIR; BioLegend), CD45-FITC, CD14-APC (Immunotools GmbH, Friesoythe, Germany) and CD15-PE (Biolegend, San Diego, CA). Aggregated and non-viable cells were excluded from the analysis. Mɸ were gated according to CD45 positive population, CD15 negative, CD14 positive and high side scatter (SSC) parameter.

To prepare a single-cell solution of heX-embryoid, the culture was treated for 45 min with Collagenase C solution (3 mg ml⁻¹ StemCell Technologies) followed by 15 min of treatment with Accuatse (Sigma). FC block solution (Thermo Fischer) was added to the samples followed by 10 min of incubation on ice. Next, the antibody mix (final dilution of 1:400) was added to the samples followed by 30 min incubation on ice. Cells were analysed using an LSR II flow cytometer (BD Bioscience) using 7-AAD (BD Pharmingen) for dead cell staining. The antibodies used were CD34-APC (Clone 581, Biolegend), KIT-BV421 (Clone 104D2, BD Bioscience), CD43-PE (Clone CD43-10G7, Biolegend), CD235ab-PE-Cy7 (Clone HIR2, Biolegend), CD33-BV605 (Clone P67.6, Biolegend), CD42b-AF700 (Clone HIP1, Biolegend), CD7-PE-Cy7 (Clone 4H9/CD7, Biolegend), CD45-Pacific Blue (Clone HI30, Biolegend), CD45-APC-Cy7 (Clone HI30, Biolegend), CD45-APC (Clone HI30, Biolegend), CD31-PE-Cy7 (Clone WM59, Biolegend), CD15-PE (Clone HI98, Biolegend), CD56-PE-Cy7 (Clone MEM-188, Biolegend) and CD49d(VLA-4)-BV605 (Clone 9F10, Biolegend). Back-gating, controls and analysis steps can be seen in Supplementary Information 1 and 2.