Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

dc.creatorSilva, Icaro Bonyek
dc.creatorNunes, Sara
dc.creatorBastos, Rana
dc.creatorLima, Reinan
dc.creatorBarbosa, Leilane
dc.creatorGrimaldi, Gabriela
dc.creatorRocha, Vinicius
dc.creatorSoares, Milena Botelho Pereira
dc.creatorVeras, Patrícia Sampaio Tavares
dc.creatorMenezes, Juliana de
dc.creatorBrodskyn, Cláudia Ida
dc.creatorTavares, Natalia
dc.date2021-12-03T13:31:28Z
dc.date2021-12-03T13:31:28Z
dc.date2021
dc.date.accessioned2023-09-26T23:49:24Z
dc.date.available2023-09-26T23:49:24Z
dc.identifierSILVA, Icaro Bonyek et al. Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response. Journal of Visualized Experiments, 2021.
dc.identifier1940-087X
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/50153
dc.identifier10.3791/62555 (2021).
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8894597
dc.descriptionFundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) under Grant number PET0009/2016 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) under Finance Code 001.
dc.descriptionMacrophages 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 106 cells per well on 24-well plates in order to obtain 2 x 105 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. Introduction
dc.formatapplication/pdf
dc.languageeng
dc.publisherMyJove Corporation
dc.rightsopen access
dc.subjectLeishmania braziliensis
dc.subjectMacrófagos
dc.subjectMonócitos
dc.subjectMicroscopia de Fluorescência
dc.subjectLeishmania braziliensis
dc.subjectMacrophages
dc.subjectMonocytes
dc.subjectMicroscopy, Fluorescence
dc.titleObtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response
dc.typeArticle


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