Figure 6.

Sorting of CD3⁺TCRαβ⁻ and CD3⁺TCRαβ⁺ MDM using flow cytometry and an experimental strategy. Human MDM were obtained after 7 days in culture, and the cells were prepared to sort CD3⁺TCRαβ⁺, CD3⁺TCRαβ⁻ and CD3⁻ MDM by flow cytometry. (A) Representative zebra plot corresponding to total MDM before (up) and after (down, left, and right) sorting. Using adequate controls, the gates were delimited to obtain MDM subpopulations, and our data showed that after sorting, we obtained at least 90% of the enriched cell subpopulation. The data are representative of six independent donors. (B) Our experimental design consisted of total and purified MDM cultured and stimulated by anti-CD3 (1 μg/mL), anti-TNF (1 μg/mL), anti-CD3 plus anti-TNF (1 μg/mL each-one) antibodies, isotype control (IgG1) antibody, negative control without stimuli (medium) and a positive control of cell activation (LPS 100 ng/mL plus IFN-γ 20 ng/mL), 24 h in culture under humid conditions (37°C and 5%CO₂). The supernatant was recovered to measure cytokines and chemokines with Luminex technology, and the cells were recovered to evaluate the gen level expression of the suppressor of cytokine signaling 3 (SOCS3) by q-PCR.