| Author | Silva, Icaro Bonyek | |
| Author | Nunes, Sara | |
| Author | Bastos, Rana | |
| Author | Lima, Reinan | |
| Author | Barbosa, Leilane | |
| Author | Grimaldi, Gabriela | |
| Author | Rocha, Vinicius | |
| Author | Soares, Milena Botelho Pereira | |
| Author | Veras, Patrícia Sampaio Tavares | |
| Author | Menezes, Juliana de | |
| Author | Brodskyn, Cláudia Ida | |
| Author | Tavares, Natalia | |
| Access date | 2021-12-03T13:31:28Z | |
| Available date | 2021-12-03T13:31:28Z | |
| Document date | 2021 | |
| Citation | SILVA, 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. | pt_BR |
| ISSN | 1940-087X | pt_BR |
| URI | https://www.arca.fiocruz.br/handle/icict/50153 | |
| Sponsorship | Fundação 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. | pt_BR |
| Language | eng | pt_BR |
| Publisher | MyJove Corporation | pt_BR |
| Rights | open access | pt_BR |
| Subject in Portuguese | Leishmania braziliensis | pt_BR |
| Subject in Portuguese | Macrófagos | pt_BR |
| Subject in Portuguese | Monócitos | pt_BR |
| Subject in Portuguese | Microscopia de Fluorescência | pt_BR |
| Title | Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response | pt_BR |
| Type | Article | pt_BR |
| Abstract | 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 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 | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Senai Institute for Innovation in Advanced Health Systems. National Service for Industrial Learning. Integrated Manufacturing and Technology Campus. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Senai Institute for Innovation in Advanced Health Systems. National Service for Industrial Learning. Integrated Manufacturing and Technology Campus. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / National Institute of Science and Technology. Research Institute in Immunology. Salvador, BA, Brazil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil / National Institute of Science and Technology. Research Institute in Immunology. Salvador, BA, Brazil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Salvador, BA, Brasil. | pt_BR |
| Subject | Leishmania braziliensis | pt_BR |
| Subject | Macrophages | pt_BR |
| Subject | Monocytes | pt_BR |
| Subject | Microscopy, Fluorescence | pt_BR |
| e-ISSN | 10.3791/62555 (2021). | |
