| Autor: |
Bonyek-Silva I; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia., Nunes S; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia., Bastos R; Oswaldo Cruz Foundation, Gonçalo Moniz Institute., Lima R; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia., Barbosa L; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia., Grimaldi G; Oswaldo Cruz Foundation, Gonçalo Moniz Institute., Rocha V; Senai Institute for Innovation in Advanced Health Systems, National Service for Industrial Learning, Integrated Manufacturing and Technology Campus, SENAI - CIMATEC Salvador., Soares MBP; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Senai Institute for Innovation in Advanced Health Systems, National Service for Industrial Learning, Integrated Manufacturing and Technology Campus, SENAI - CIMATEC Salvador., Veras PST; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; National Institute of Science and Technology (INCT), Research Institute in Immunology., de Menezes J; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia., Brodskyn C; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia; National Institute of Science and Technology (INCT), Research Institute in Immunology., Tavares N; Oswaldo Cruz Foundation, Gonçalo Moniz Institute; Federal University of Bahia; natalia.tavares@fiocruz.br. |
| Abstrakt: |
Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 10 6 cells per well on 24-well plates in order to obtain 2 x 10 5 macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species. |
