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. 2022 Oct 18;11:e77373. doi: 10.7554/eLife.77373

Figure 2. Validation of macrophage polarisation and metabolic profiling of IFNγ-M1, IL-4-M2, and untreated (UT) macrophages.

(A, B) ELISA of inflammatory cytokine TNFα and anti-inflammatory IL-10 in IFNγ-M1, IL-4-M2, and UT macrophages. (C–F) Evaluation of CXCL9, MRC1, CXCL10, and CCL13 gene expression in IFNγ-M1, IL-4-M2, and UT macrophages. (G,J ) Extracellular acidification ratio (ECAR) and oxygen consumption ratio (OCR) profile of IFNγ-M1, IL-4-M2, and UT macrophages when treated sequentially with oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone + antimycin A, and 2-deoxy-d-glucose (2-DG). (H, I, K, L) Area under the curve (AUC) values calculated from ECAR and OCR between each treatment. Data displayed as average ± SD. Statistical significance verified by one-way ANOVA with *p<0.05, **p<0.01, ***p<0.001 to show significance for N = 6 donors.

Figure 2—source data 1. ELISA, gene expression, extracellular acidification ratio, and oxygen consumption ratio measurements for each replicate data and details of statistical tests and chosen parameters.

Figure 2.

Figure 2—figure supplement 1. Validation of macrophage polarisation using flow cytometry.

Figure 2—figure supplement 1.

Primary human macrophages were left untreated (UT), treated with IFNγ (20 ng/ml), or IL-4 (20 ng/ml) for 24 hr. Cells were stained for M1 maturation surface markers CD80, CD86, and M2 surface markers CD163, CD206, and analysed by flow cytometry. (A–D) Representative histograms depicting median fluorescence intensity (MFI) of surface markers. Bar graphs depict MFI as a percentage to the control (untreated cells) (N = 3). All data is represented as mean ± SEM and analysed by one-way ANOVA with Dunnett’s multiple-comparisons test.
Figure 2—figure supplement 1—source data 1. Flow cytometry surface markers measurement and details of statistical tests and chosen parameters.