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. 2019 Jul 2;27(9):1597–1611. doi: 10.1016/j.ymthe.2019.06.010

Figure 3.

Figure 3

Functional Characterization of Csf2ra Gene-Corrected Macrophages before PMT

(A) GM-CSF clearance assay. Vector-mediated Csf2ra expression in Csf2ra−/− BMDMs restores clearance of GM-CSF from culture medium. Groups include BMDMs from wild-type mice (WT), Csf2ra−/− mice before or after mCsf2ra-LV transduction (Csf2ra−/− or Csf2ra−/− + mCsf2ra-LV, respectively), and medium without cells (No cells). Data indicate mean ± SD of 3 independent LSK-derived macrophage preparations and transductions per condition at each time point. (B) STAT5 phosphorylation assay showing that Csf2ra-LV transduction restores GM-CSF signaling in Csf2ra−/− BMDMs. (C) Phagocytosis assay. Flow-cytometry-based quantification of phagocytic internalization of opsonized latex beads. Histogram peaks showing internalization of 1, 2, 3, etc., beads per cell (indicated) by Csf2ra−/− BMDMs before and after mCsf2ra-LV transduction and culture in medium containing GM-CSF. The percentage of cells containing internalized latex beads is indicated for each condition (n = 3). (D) Surfactant clearance assay (n = 3). The ability of BMDMs (non-transduced WT or Csr2ra−/−, or mCsf2ra-LV-transduced Csf2ra−/−) to clear surfactant was evaluated by measuring surfactant-derived cholesterol content before or immediately after a 24-h “pulse” exposure to surfactant, or after pulse exposure, washing to remove extracellular surfactant, and culture for 24 h to permit clearance of surfactant-derived cholesterol (indicated). *p < 0.05; **p < 0.01; ns, not significant.