Cell surface marker antibodies

Cells from air-pouch and BALFs were stained with various cell surface marker antibodies from BD Biosciences (San Diego, CA) or Biolegend (San Diego, CA). Data were acquired on FACS Calibur or FACS Canto (BD Biosciences) and analyzed using Flowjo software (Tree Star). In lungs leukocytes were identified as CD45⁺ cells, macrophages as CD45⁺ CD11c^hi F4/80⁺ cells, eosinophils as CD45⁺ CD11c^lo Siglec-F⁺ cells, neutrophils as CD45⁺ CD11c^lo Siglec-F⁻ Ly6G^hi cells. In scatter-gated cells of air-pouch lavage fluids, leukocytes were identified as CD45⁺ cells, neutrophils as Ly6G^hi cells, eosinophils as Ly6G^lo Siglec-F⁺ cells, macrophages as Ly6G^lo Siglec-F⁻ F4/80⁺ cells. Percentage of various cell types thus obtained were multiplied to the total cell counts for actual cell numbers of neutrophils, eosinophils, and macrophages.

In order to verify that cells we extracted were BMSCs, we detected the expression of cell surface markers by flow cytometry. Briefly, we collected cells at the second passage and resuspended them in PBS. Then, 2 × 10⁵ cells were incubated with PE or FITC labeled cell surface marker antibodies against CD29, CD34, CD44 and CD45 (BD Biosciences, USA), as well as isotype control antibody at 4°C for 30 minutes in the dark. After the incubation is completed, the samples were analyzed by the flow cytometer (BD Biosciences, USA).