INTRODUCTION: Genetic association studies in complex diseases often describe genes and variants that need to be further investigated for their functional role. One of the strategies includes the use of in vitro cell models. OBJECTIVES: This study aims to set up an optimized panel of in vitro functional assays using Monocyte-derived Macrophages stimulated with immune response activators and specific antigens for functional parameters. MATERIALS AND METHODS: Initially, the project proposed the use of THP-1 cells grown in vitro. However, adaptations were necessary, and it was decided to replace the macrophage-like cells produced from THP-1 by the monocytederived macrophages (MDM) isolated from peripheral blood. For the isolation of peripheral blood mononuclear cells, the principle of density gradient separation with Histopaque 1077 Hybri-Max was used. The morphological differentiation of monocytes into macrophages was performed by testing different concentrations of M-CSF. Cell differentiation was evaluated by morphological criteria and expression of the cell membrane marker CD14. Cells were submitted to classic macrophage activators: Interferon-γ and LPS or BCG MOI 50:1 at different concentrations. The evaluation of the expression of cell surface molecules and the functional assays were conducted by flow cytometry on a FACSCalibur platform or through the Cytell™ Cell Imaging System. The evaluation of cell viability was carried out by the evaluation of the necrosis index by the dye 7-AAD, and apoptosis by the marker Annexin V. To evaluate the ability to present antigens by the MDM, the markers FITC-CD80, PE-CD86 and APCCD54 were used. The assessment of the ability of macrophages to produce reactive oxygen species (ROS) was performed using the DHE indicator, a fluorogenic dye specific for superoxide and hydrogen peroxide. Cell acquisition was performed using the FACSCalibur flow cytometer using the CellQuest software. The analysis of the data obtained in the cytometry was performed using the Flow-Jo software. RESULTS: Macrophage immunophenotyping was confirmed by the titrated CD14 marker. The volume of 10μl was considered the best when evaluating cost/effectiveness. Based on the concentrations of LPS and IFN-γ tested, activation with 200ng/mL of LPS and 2,000 U/mL of IFN-γ was considered ideal. Cell marking using 5μl of CD54 performed well when compared with 10μl and 20μl of the same marker. For late apoptosis (7- AAD) and early apoptosis (Annexin V) analysis, three volumes of the marker and dye were tested; the best result was observed with the volume of 2μl for both reagents. For the evaluation of ROS production, the ideal concentrations of DHE and H2O2 were 200 μM and 10 μM, respectively. FINAL CONSIDERATIONS: The methodologies introduced in our laboratory from this study will allow the implementation of functional studies involving different situations of investigation of leprosy and other infectious diseases.
