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.
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.
The CNS is mainly composed of neurons and glial cells1,2,3. 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 CNS3,4, 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 neurons6,7, emerging functions of these cells have been described in the recent literature, including their responses to infections8,9,10,11. Thus, there is a push to develop methods to isolate these glial cells to understand their functions individually.
There are some alternative models to study glial cells rather than primary cultures, like immortalized cell lineages and in vivo models. However, immortalized cells are more likely to undergo genetic drifting and morphological changes, while in vivo studies impose limited manipulation conditions. Conversely, primary cultures are easy to handle, better resemble in vivo cells and also allow us to control experimental factors12,13. Here, we describe guidelines on how to extract, maintain and dissociate murine astrocytes and microglia primary cells in the same protocol. Furthermore, we also provide examples on how to work with protozoa infection in these cultures.
CNS cells extracted from neonatal mice (up to 3 day-old) were cultured for 14 days on differential media that allows the preferential growth of astrocytes and microglia cells. Since microglia rest above the attached astrocytes, cell populations were mechanically dissociated in an orbital incubator. Next, we collected all the supernatant containing microglia and added trypsin to detach astrocytes. Isolated glial cells were phenotypically evaluated by flow cytometry and plated according to the desired experiment.
We also provided examples on how to infect these isolated microglia and astrocytes with protozoa parasites. T. gondii is a highly neurotropic protozoan responsible for toxoplasmosis14, while T. cruzi is responsible for Chagas disease which can leads to development of neurological disorders in the CNS15,16. Furthermore, it has also been reported that infection with T. gondii17,18 or T. cruzi19,20,21 were the presumable cause of death in immunocompromised patients. Therefore, the elucidation of immunologic role of glial cells from the CNS in controlling protozoa infections is of great importance.
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 2.7 x 107 astrocytes and 4 x 106 microglia were obtained from six neonatal C57BL/6 mice. All procedures were performed under sterile condition in a class II biosafety cabinet.
2. Infection and Evaluation of Infection Rates
On the 14th day, glial cells culture (Figure 1A) underwent mechanical dissociation. Isolated cell populations were analyzed by flow cytometry according to CD11b, CD45 and GFAP markers. We could observe a purity of 89.5% for the astrocyte population and 96.6% for the microglia population (Figure 1B). After isolation, cells were plated in a 96-well flat plate and after 24 h they were ready to be infected by T. cruzi or T. gondii according to the respective infection protocols. Here we provided a time-course infection of T. cruzi as an example of infection rate evaluation in these glial cells.
Astrocytes and microglia (3 x 104 cells/well) were infected with T. cruzi at MOI = 5:1 (parasites:cell) for 2–96 h (Figure 2). Infection rate was evaluated by immunofluorescence by counting the number of cells and the number of parasites stained with a DNA intercalator (DAPI). Briefly, we could observe that T. cruzi is able to invade microglia and astrocytes at similar rates. Moreover, T. cruzi replicates in both cell types, reaching the highest infection rate at 96 h post infection (p.i.). Interestingly, at 96 h p.i. the infection rate is more pronounced in astrocytes than in microglia, suggesting that microglia cells are able to control the parasite replication more efficiently than astrocytes, as we previously described11.
It is important to note that the use of genetically modified parasites expressing fluorescent reporters or parasites labeled with specific fluorescent antibodies could improve the immunofluorescence microscopy, since they are better distinguished from cell nucleus (Figure 3). Moreover, the infection rate of fluorescent parasites can be determined by other techniques such as flow cytometry. Here we provided examples of T. gondii RH strain constitutively expressing YFP (Figure 3A) and T. cruzi Y strain stained with non-commercial mAb 2C2 anti-Ssp-4 protein (Figure 3B).
Altogether, this protocol describes how to extract, maintain, dissociate and infect murine microglia and astrocytes primary cells, which could be a powerful tool to study, for example, immune responses of glial cells to protozoa infection as briefly elucidated here.
Figure 1: Primary astrocytes and microglia cells. (A) Images of C57BL/6 murine glial cells on the 14th day of culture were obtained by inverted microscope (400x). The red arrow indicates the layer of astrocytes and the black arrow indicates the microglia. (B) After mechanical dissociation, astrocytes and microglia were stained (5 x 105 cells) with fluorescent anti-CD11b, anti-CD45 and anti-GFAP and evaluated by flow cytometry (1 x 105 cells acquisition). Astrocytes are considered as CD11b–/CD45– and GFAP+ cells, whereas microglia are CD11b+/CD45+ and GFAP– cells. Please click here to view a larger version of this figure.
Figure 2: Time-course of T. cruzi infection in glial cells. Astrocytes and microglia from C57BL/6 mice were infected with T. cruzi Y (MOI = 5:1). After 2 h, 48 h and 96 h, chambers were fixed with methanol and stained with cell nucleus marker DAPI (blue) and anti-GFAP (red). Images were obtained by inverted fluorescence microscope (400x). The number of amastigotes inside the cells and the frequency of infected cells were evaluated by the fluorescence microscope software (see Table of Materials). Please click here to view a larger version of this figure.
Figure 3: Astrocytes and microglia infection with fluorescent protozoan parasites. (A) 3 x 104 astrocytes and microglia from C57BL/6 mice were infected (MOI = 1:1) with T. gondii RH YFP (green) for 48 h, chambers were fixed with 1% PFA and stained with DAPI (blue). (B) 3 x 104 astrocytes and microglia from C57BL/6 mice were infected (MOI = 5:1) with T. cruzi Y for 96 h, and chambers were fixed with pure methanol and stained with DAPI (green) and mAb 2C2 anti-Ssp-4 (orange). Images were acquired using an inverted fluorescence microscope. Please click here to view a larger version of this figure.
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 conditions8,9,24. 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 energy supply, neurometabolism, immune surveillance, synaptic pruning, shaping and modulation of the tissue, among others3,25,26,27,28,29.
Since astrocytes and microglia can be differentially modulated during infection or sterile inflammation, it is of great importance to understand the individual and relative role of these glial cells. Although microglia are known to represent the mononuclear phagocyte system (MPS) in CNS, astrocytes have also been described as a pivotal player in the CNS immune responses8. In order to compare the role of each glial subset in the context of infection and/or inflammation, it is mandatory to obtain them from the same extraction and conditions. There are few reports about the effector responses of microglia and astrocytes to control infections, especially in regard to protozoan parasites. In this sense, we recently demonstrated the requirement of NLRP3 inflammasome to the control of T. cruzi replication in microglia but not in astrocytes by a mechanism involving nitric oxide (NO) secretion11.
This protocol describes a method that is focused on simultaneously extracting the two most abundant glial cells populations, astrocytes and microglia, from postnatal (up to 3-day-old) mice. Newborn mice older than 3-day-old can be used, but we experienced that the yield of both cells decreases slightly over time (data not shown). Our method differs from previously described protocols for astrocytes or microglia isolation because we focused on obtaining and isolating both cell types in the same extraction, under the same conditions, thus optimizing the usage of experimental animals and improving the study of the relative role for those cells in immunological contexts. Moreover, our protocol also optimized the yield of both cell types. The yield is usually 3–5 x 106 astrocytes and 3–5 x 105 microglia per flask. Compare this to other protocols that result in less cells using more animals per T-75 flask (some protocols use 2–3 animals per flask)30,31.
Another advantage of this protocol is the time required to obtain isolated astrocytes and microglia. Within 14 days it is possible to obtain mature microglia. In fact, it is important to highlight that, as Flode and Combs demonstrated, microglia older than 14 days present diminished ability to secrete cytokines; it is very important to consider this in neuroimmunological contexts30. Despite the fact that the mature makers for astrocyte are not completely understood, GFAP is largely used to characterize responsive and active astrocytes. Some protocols achieve levels of 90% GFAP positive cells only after 21 or 28 days of culture. For that reason, we proposed a method that provided, cells that are immunoresponsive and mostly mature within 7–14 days. Although thepurity can be lower than other methods for both astrocyte31 and microglia30, the main focus was to obtain microglia and astrocytes in greater quantities in a concomitant extraction. We understand that cell populations could be further sorted or isolated with column separation to improve their purities. For this, additional cell markers might be included, such as Iba1 or TMEM119 for microglia; Aquaporin-4 or S100B for astrocytes, CC1 or O4 for oligodendrocytes and NeuN for neurons22,31,32,33. However, an important cell loss at the end of the protocol should be considered.
Thus, we provided a modified method to concomitantly obtain microglia and astrocytes in an efficient and cheap protocol. Moreover, this protocol provide the advantages of a great yield allowing the comparative studies of glial subsets in neuroimmunological contexts, as illustrated here during T. cruzi and T. gondii infection.
The authors have nothing to disclose.
We would like to thank professor Dr. Renato A. Mortara from Federal University of São Paulo (UNIFESP) for mAb 2C2 anti-Ssp-4. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 2017/25942-0 to K.R.B.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 402100/2016-6 to K.R.B.), Instituto Nacional de Ciência e Tecnologia de Vacinas (INCTV/CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001). M.P.A. receives fellowship from CNPq, A.L.O.P. receives fellowship from CAPES, and I.S.F and L.Z.M.F.B. receives fellowship from FAPESP.
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 60×15 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 |