Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Author(s)

Lima Pacheco, Aline de Oliveira ; Amaral, Marcelo Pires ; de Farias, Ingrid Sancho ; Miguel Fahur Bottino, Luiza Zainotti ; Bortoluci, Karina Ramalho

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 157, p. 8-pg., 2020-03-01.

Abstract

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis. (AU)

• FAPESP's process: 17/25942-0 - Cross talk between RIP3K and NLRP3 during T. cruzi infection
• Grantee: Karina Ramalho Bortoluci
• Support Opportunities: Regular Research Grants