Figure 4.

Validation of the method of sorting CD45⁺empty PE⁻ CD11b^highCD11c^intermediateGr‐1^intermediatecells by flow cytometry. (A) The gating strategy for the collection of more purified CD45⁺ GFP⁺ cells. Approximately 80% of CD45⁺ empty PE⁻ CD11b^high CD11c^intermediate Gr‐1^intermediate cells were GFP⁺ in the Col‐GFP reporter strain on day 7 after BLM treatment (bottom right panel). One representative experiment from a total of three repeats with a total of six mice is shown. (B) The expression of collagen I(α)1 and collagen I(α)2 in sorted CD45⁺ empty PE⁻ CD11b^high CD11c^intermediate Gr‐1^intermediate GFP⁺cells from the lungs were examined by RT‐PCR. (C) The phenotype of fibrocytes in the lungs of the Col‐GFP reporter strain treated with BLM. The lungs treated with BLM were harvested on Day 7. Single cell suspensions were collected from the minced lungs. The cells were then cultured on dishes and adherent cells were harvested. Flow cytometry was performed with GFP⁺ adherent cells using the gating strategy shown in (A). One representative experiment from a total of three repeats with a total of six mice is shown. BLM, bleomycin; GFP, green fluorescent protein; RT‐PCR, reverse‐transcription polymerase chain reaction