Key Role of STAT4 Deficiency in the Hematopoietic Compartment in Insulin Resistance and Adipose Tissue Inflammation

Figure 3

Immune cell composition of the SVF of epididymal adipose tissue of chimeric mice on HFD. (a) SVF was stained with fluorescently labeled anti-mouse CD45, CD3, CD4, and CD8 antibodies and analyzed by flow cytometry. Cell number was calculated based on the corresponding antibody positivity. To determine different T cell subsets, cells in the CD45 positive gate were analyzed. Data represent mean ± SD from mice/group. (b) Representative plots showing the general strategy for the CD45+ gating and for CD3 positivity and double positivity for CD3CD4 and CD3CD8 within the CD45+ gate of each group. (c) Flow cytometry analysis of SVF stained for macrophage markers CD11b and F4/80 and the APC marker CD11c. Data represents average ± SD of 5 mice. The number of positive cells was determined within the CD45+ gate. (d) Representative immunofluorescent micrographs showing localization of STAT4 (red) and Mac-2 (green) in epididymal adipose tissue of STAT4+/+ mice (i–iii) and STAT4−/− mice (iv–vi); (vii) shows a crown-like structure containing some cells immunopositive for both Mac-2 and STAT4 (arrows); (viii) shows the nonimmune IgG control; magnification 200x for (i–iii), (iv–vi), and (viii); 400x for (vii). CD11b+ and CD3+ cells were isolated from adipose tissue of chimeric mice by immunoseparation and DNA was extracted and analyzed for presence of STAT4 gene following amplification with specific primers and separation on agarose gels. Band densitometry is also showed and indicates presence of both STAT4-sufficient and STAT4-deficient CD3+ and CD11b+ cells in adipose tissue of both chimeras.

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