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. 2022 Mar 8;7(5):e150239. doi: 10.1172/jci.insight.150239

Figure 4. IFN-γ is sufficient to remodel and protect macrophages.

Figure 4

(A–C) RAW264.7 cells were treated with recombinant mouse IFN-γ or vehicle prior to being infected with Sp3 for 2 hours. (A) Infected cells were stained with Hoechst (nucleus, blue), Cell Mask Green (plasma membrane, green) and Nuclear Red Dye 647 (permeable dye, red). Scale bar: 100 μm. (B) Quantification of cells with permeable plasma membranes, defined by nuclear red staining. n = 3 independent experiments. (C) Quantification of ell death, as measured by LDH in the supernatant. n = 5 independent experiments. Paired t test analyses were used to examine significance. (D–F) Seven- to 8-week-old mice were i.t. stimulated with IFN-γ or vehicle (1% BSA in saline) directed to the left lung twice at a 1-week interval, to mimic infection experiences. Four weeks after the second stimulation with IFN-γ, single-cell suspensions were created from left lung lobes and processed using flow cytometry. AM were identified as CD11c+SiglecF+CD64+ cells. n = 9–10 mice per group. (D) AM numbers were calculated from the frequency of total measured by FlowJo software. (E and F) Median fluorescent intensity (MFI) of Siglec F and MHC-II expression on AM. Values are expressed as mean ± SEM. Unpaired t test analyses were used to examine significance.