Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain

Figures

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  Figure 1. Detection of CD45+ GFP+ cells in the lungs of the Col‐GFP reporter strain. (A) Total lung cells from adult wild‐type mice and the Col‐GFP reporter strain were isolated enzymatically and the expression of GFP together with CD45 (APC/Cy7) was evaluated by flow cytometry. (B) FACS profiles from CD45+ cells isolated from the same wild‐type mice and the Col‐GFP reporter strain are shown at the upper panel. After gating out F4/80 (APC)+ empty PE+ cells (P2 fraction), GFP+ cells (P1 fraction) were present in the Col‐GFP reporter strain (lower right). (C) The surface phenotype of gated out F4/80+ empty PE+ cells was examined by flow cytometry. Those cells were stained with CD64 PECy7, MerTK PECy7, CD11b PECy7, CD11c PECy7 antibodies or each isotype control antibody. Relative expression of each molecule is shown in histograms as blue lines overlaid with negative controls (red lines). One representative experiment from a total of three repeats with a total of six mice per group is shown. GFP, green fluorescent protein

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  Figure 2. The surface phenotype of CD45+ GFP+ cells in the lungs of the BLM‐treated Col‐GFP reporter strain. (A) The increase of CD45+ GFP+ lung cells from the Col‐GFP reporter strain upon treatment with BLM was monitored at 7 (upper right), 14 (lower left), and 21 days (lower right) after intratracheal instillation. (B) The surface phenotype of CD45+ GFP+ cells on Day 7 after BLM treatment was examined by flow cytometry. Relative expression of each molecule is shown in the histogram as green lines overlaid with negative controls (red lines). One representative experiment from a total of three repeats with a total of six mice per group is shown. BLM, bleomycin; GFP, green fluorescent protein

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  Figure 3. The characteristic expression pattern of cell adhesion molecules in CD45+ GFP+ cells. (A) The expression profiles of CD11b, CD11c, Gr‐1, Ly6C, and Ly6G in CD45+ GFP+ cells on Day 7 after BLM treatment (upper panels) were compared with CD45+ empty PE+ cells without BLM treatment (lower panels) by flow cytometry. (B) FACS profiles from CD45+ GFP+ cells isolated from the Col‐GFP reporter strain with (right panels, on Day 7) or without BLM treatment (left panels) are shown. One representative experiment from a total of three repeats with a total of six mice is shown. BLM, bleomycin; GFP, green fluorescent protein

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  Figure 4. Validation of the method of sorting CD45+empty PE− CD11bhighCD11cintermediateGr‐1intermediatecells by flow cytometry. (A) The gating strategy for the collection of more purified CD45+ GFP+ cells. Approximately 80% of CD45+ empty PE− CD11bhigh CD11cintermediate Gr‐1intermediate 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− CD11bhigh CD11cintermediate Gr‐1intermediate 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…