Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Summary

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

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.

Introduction

The CNS is mainly composed of neurons and glial cells^1,2,3. Microglia and astrocytes are the most abundant glia cells in the CNS. Microglia, the resident macrophage, is the immunocompetent and the phagocytic glia cell in the CNS^3,4, while astrocytes are responsible for maintaining homeostasis and exert supportive functions⁵.

Despite glial cells being classically known to be responsible for the support and protection of neurons^6,

Protocol

All experimental procedures involving mice were carried out in accordance to the Brazilian National Law (11.794/2008) and approved by the Institutional Animal Care and Use Committees (IACUC) of the Federal University of São Paulo (UNIFESP).

1. Glial Cells Extraction, Maintenance and Dissociation

NOTE: The number of mice used for the glial cells extraction depends on the quantity of cells required to perform the desired experiments. In this protocol, a total of 2........

Discussion

The importance of studying isolated glial cells functions in distinct biological contexts has been expanding in the last two decades. Understanding the CNS beyond neurons is still a growing field in cell biology, especially under infections or inflammatory conditions^8,9,24. Glial cells are crucial not only for neurons physical support (as it was previously known), but also in many other physiological situations such as neuron en.......

Materials

Name	Company	Catalog Number	Comments
70% Ethanol	Dinâmica Química Contemporânea	Cat: 2231	Sterilize
75 cm2 Flask	Corning	Cat: 430720U	Plastic material
96 well cell culture plate	Greiner Cellstar	Cat: 655090	Cell culture
Ammonium Chloride (NH4Cl)	Dinâmica Química Contemporânea	Cat: C10337.01.AH	Remove autofluorescence
Anti-GFAP antibody	Abcam	Cat.: ab49874	Immunofluorescence antidoby
Bottle Top Filter 0.22 mm CA	Corning	Cat: 430513	Culture medium filter
Bovine Serum Albumin (BSA)	Sigma Aldrich	Cat: A7906	FACS Buffer preparation
CD11b (FITC)	BD Pharmigen	Cat.: 553310	Flow cytometry antibody
CD45 (PE)	Invitrogen	Cat.: 12-0451-83	Flow cytometry antibody
Centrifuge	Eppendorf	Cat: 5810R	Centrifugation
Centrifuge	Eppendorf	5415R	Centrifugation
Class II biosafety cabinet	Pachane	Cat: 200	Biosafety cabinet for sterile procedures
CO2 Incubator	ThermoScientific	Model: 3110	Primary cells maintenance
Conical tubes 15 mL	Corning	Cat: 430766	Plastic material
Conical tubes 50 mL	Corning	Cat: 352070	Plastic material
Countess automated cell counter	Invitrogen	Cat: C10281	Cell counter
DAPI	Invitrogen	Cat.: D1306	Immunofluorescence antidoby
Digital Microscope Camera	Nikon	Cat: DS-RI1	Capture images on microscope
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	Cat: 12800-058	Cell culture medium
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	Cat: E9884	FACS Buffer preparation
F12 Nutrient Mixture	Gibco	Cat: 21700-026	Cell culture medium
FACS Canto II	BD Biosciences	Unavaiable	Flow cytometer
Fetal Bovine Serum (FBS)	LGC Biotechnology	Cat: 10-bio500-1	Cell culture medium supplement
Flow Jo (software)	Flow Jo	Version: Flow Jo_9.9.4	Data analysis
Fluorescence intenselight	Nikon	Cat: C-HGFI	Fluorescence source
GFAP (APC)	Invitrogen	Cat.: 50-9892-82	Flow cytometry antibody
Goat - anti-mouse IgG (FITC)	Kirkeegood&Perry Lab (KPL)	Cat.: 172-1806	Immunofluorescence antidoby
HBSS - Hank's Balanced Salt Solution	Gibco	Cat: 14175079	Cell culture medium
HEPES	Sigma Aldrich	Cat: H4034	Cell culture medium supplement
IC Fixation Buffer	Invitrogen	Cat: 00-8222-49	Cell fixation for Flow Citometry
Inverted microscope	Nikon	Model: ECLIPSE TS100	Microscope
Isoflurane	Cristália	Cat: 21.2665	Inhaled anesthetic
Methanol	Synth	Cat: 01A1085.01.BJ	Fixation for Immunofluorescence
Micro spatula	ABC stainless	Unavaiable	Surgical material
Microtube 1.5 mL	Axygen	Cat: MCT-150-C	Plastic material
Monoclonal antibody (mAb) 2C2 anti-Ssp-4	Non commercial	Non commercial	Immunofluorescence antidoby
Multichannel Pipette (p200)	Corning	Cat: 751630124	Pipette reagents
NIS Elements Software	Nikon	Version 4.0	Acquire and analyse images
Non-fat milk	Nestlé	Cat: 9442405	Blocking solution for immunofluorescence
Orbital Shaker Incubator	ThermoScientific	Model: 481 Cat: 11	Dissociate microglia from astrocytes
Paraformaldehyde (PFA)	Sigma Aldrich	Cat: P6148	Fixation for Immunofluorescence
PBS	Non commercial	Non commercial	Neutral Buffer
Penicillin G	Sigma Aldrich	Cat: P-7794	Cell culture medium supplement
Permeabilization Buffer (10X)	Invitrogen	Cat: 00-8333-56	Cell permeabilization for Flow Citometry
Petri dish 60x15 mm (Disposable, sterile)	Prolab	Cat: 0303-8	Plastic material
pH meter	Kasvi	K39-1014B	Calibrate pH solution
RPMI 1640 Medium	Gibco	Cat: 31800-014	Cell culture medium
Scissors	ABC stainless	Cat: LO9-W4	Surgical material
Serological pipette 10 mL	Corning	Cat: 4101	Plastic material
Serological pipette 5 mL	Corning	Cat: 4051	Plastic material
Single Channel Pipette (p1000)	Gilson Pipetman	Cat: F123602	Pipette reagents
Single Channel Pipette (p200)	Gilson Pipetman	Cat: F123601	Pipette reagents
Sodium bicarbonate	Sigma Aldrich	Cat: S6297	Cell culture medium supplement
Streptomycin sulfate salt	Sigma Aldrich	Cat: S9137	Cell culture medium supplement
Triton X-100	Sigma Aldrich	Cat: T9284	Permeabilization for immunofluorescence
Trypsin	Gibco	Cat: 27250-018	Digestive enzyme
Tweezers	ABC stainless	Cat: L28-P4-172	Surgical material
Water Bath	Novatecnica	Model: 09020095	Digeste tissue at 37 ºC with trypsin