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^6,7, emerging functions of these cells have been described in the recent literature, including their responses to infections^8,9,10,11. 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 factors^12,13. 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 CNS^15,16. Furthermore, it has also been reported that infection with T. gondii^17,18 or T. cruzi^19,20,21 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.

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 2.7 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.

1. Day 1
   1. Prepare pure Hank's balanced salt solution (HBSS) and HBSS + 10% of heat-inactivated fetal bovine serum (FBS) (see Table of Materials).
   2. Prepare supplemented Dulbecco's Modified Eagle Medium (DMEM)/F12 (10% FBS + 0.08 mM Penicillin + 0.09 mM Streptomycin + 12.5 mM HEPES + 30 mM sodium bicarbonate, pH 7.2) and filter it with a 0.22 µm filter (see Table of Materials).
      NOTE: All culture media should be stored at 4 °C, but it is necessary to prewarm them at 37 °C for 20 min before starting the experiment.
   3. Sterilize all surgical instruments (scissors, spatula and tweezers) in an autoclave and use 70% ethanol during the procedure.
   4. Put newborns (up to 3 day-old) mice in a sealed chamber containing cotton soaked with isoflurane for 5 min for profound anesthesia.
   5. Spray the mouse pup with 70% ethanol and decapitate the animal with scissors.
   6. Make sagittal cut (posterior to anterior) with scissors along the cranium to open it and expose the brain. Separate the skull from the brain using tweezers. Remove the brain using a micro spatula to maintain the brain integrity.
   7. Place the brain in a dry Petri dish (6 cm diameter). Remove the olfactory bulb and cerebellum using a micro spatula. Move the cortex to another Petri dish (6 cm diameter) containing HBSS + 10% FBS (2 mL/Petri dish).
   8. Cut the brain tissue into small pieces with sterile scissors and using a p1000 micropipette transfer each brain tissue with HBSS + 10% FBS to different 15 mL conical tubes. Make sure that the final volume is 2 mL/tube. If not, complete with HBSS + 10% FBS.
      NOTE: The protocol can be paused here. If so, conical tubes with CNS tissue must be placed on ice up to 1 h.
   9. Wash the cut brain tissue with 3 mL of HBSS + 10% FBS (per tube) and after decantation. Carefully remove the supernatant. Repeat this step 3 times.
      NOTE: This step aims to remove the debris and to recover the extracted tissue.
   10. Wash the cut brain tissue with 3 mL of pure HBSS (per tube) and after decantation, carefully remove the supernatant. Repeat this step 3 times.
       NOTE: This step aims to remove the remaining serum from the previous washes, as cells will be trypsinized in the next step.
   11. Add 3 mL of trypsin per tube and place in a water bath at 37 °C for 30 min, gently shaking the tubes every 5 min. Avoid bubbles by inverting or abruptly mixing the tubes.
       NOTE: This step aims to digest the collected tissue.
   12. To inactivate the trypsin, wash the tissue with 3 mL per tube of HBSS + 10% FBS and, after decantation of the tissue, carefully remove the supernatant. Repeat this step 3 times.
   13. Wash the tissue with 3 mL per tube of pure HBSS and carefully remove the supernatant. Repeat this step 2 times.
   14. Add 7 mL per tube of pure HBSS and homogenize the tissue through successive passages in pipettes: first with the 10 mL serological pipette, followed by the 5 mL and finally with the p1000 micropipette.
   15. After homogenization, centrifuge tubes at 450 x g for 5 min at 4 °C.
   16. Discard the supernatant and resuspend the pellet with 4 mL of HBSS + 10% FBS per tube.
   17. Transfer cells to a pretreated T-75 flask for optimal cell culture adhesion (see Table of Materials).
       NOTE: Process tissue from each animal per flask.
   18. Place the flask in the incubator at 37 °C and 5% CO₂ for 30 min for adherence.
   19. Add 10 mL of supplemented DMEM/F12 per flask and incubate at 37 °C and 5% CO₂.
       NOTE: The final volume is 14 mL per flask.
2. Day 3
   1. Remove 7 mL of culture medium from the T-75 flask and add 7 mL of fresh supplemented DMEM/F12.
      NOTE: The flasks will contain a lot of cellular debris and a cloudy appearance.
3. Day 5
   1. Remove all the medium from the T-75 flask and add 14 mL of fresh supplemented DMEM/F12.
      NOTE: Medium is replaced from the flasks to remove debris and non-adherent cells.
   2. After each 48 h, remove 6 mL of the supernatant and add 7 mL of fresh supplemented DMEM/F12 medium until day 14 of culture.
4. Day 14
   1. After removing 6 mL of the supernatant and adding 7 mL of fresh supplemented DMEM/F12 medium, close the T-75 flasks tightly.
   2. Place them in the floor orbital shaker at 200 rpm and 37 °C overnight to mechanically dissociate microglia from astrocytes.
5. Day 15
   1. Take the flasks from the shaker and vigorously wash them with their own medium in order to optimize cell dissociation and harvest the maximum number of microglia. Then, collect the supernatant (containing microglia) and transfer to a 50 mL conical tube.
   2. Next, add 4 mL of trypsin into each flask and incubate for 5 min at 37 °C in order to detach the astrocytes.
   3. Add 5 mL per flask of supplemented DMEM/F12 to inactivate the trypsin. Wash the flasks with their own medium and transfer contents to a 50 mL conical tube.
   4. Centrifuge all tubes containing dissociated microglia and astrocytes at 450 x g for 5 min at 4 °C.
   5. Discard the supernatant. Resuspend the microglia pellet in 1 mL and the astrocytes in 10 mL of supplemented DMEM/F12.
   6. Proceed to cell counting in a Neubauer chamber or an automatic cell counter. Plate the cells with supplemented DMEM/F12 at the desired density in an appropriate flat bottom cell culture plate (pretreated for optimal cell attachment; see Table of Materials). Incubate cells at 37 °C and 5% CO₂ for 24 h to allow them to attach.
      NOTE: Reserve 1.5 x 10⁶ of each cell population to confirm their purity by flow cytometry.
   7. Perform a flow cytometric analysis to verify the purity of cell populations.
      NOTE: We consider microglia CD11b⁺/CD45⁺/GFAP^- and astrocytes CD11b^-/CD45^-/GFAP⁺. Iba1 and TMEM119 staining might be considered in order to fully characterize microglia²².
   8. Add a FMO (Fluorescence Minus One)²³ and an unstained sample for each cell population (astrocytes and microglia) as controls of flow cytometry assay.
   9. For each cell population, reserve 5 x 10⁵ cells for each stained and unstained samples. For FMO, reserve two other tubes of microglia containing 2.5 x 10⁵ cells each and also reserve another tube of 5 x 10⁵ astrocytes.
      NOTE: Since microglia numbers can be a limiting factor, it is possible to use fewer cells for unstained and FMO controls.
   10. Centrifuge all microtubes at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the pellet with 500 µL/microtube of FACS buffer (phosphate-buffered saline (PBS) + 0.5% bovine serum albumin (BSA) + 2 mM EDTA, pH 7.2).
   11. Centrifuge all microtubes at 450 x g at 4 °C for 5 min. Discard the supernatant and resuspend the pellet with 100 µL/microtube of anti-CD16/CD32 (clone 93) (1:50) diluted in FACS buffer. Incubate for 10 min at room temperature.
   12. Add 500 µL/microtube of FACS buffer into all microtubes and centrifuge at 450 x g at 4 °C for 5 min. Discard the supernatant. Resuspend astrocytes and microglia staining tubes and also astrocyte FMO tube with 50 µL/microtube of surface markers mix: anti-CD45 (PE, clone 30-F11) and anti-CD11b (eFluor 450, clone M1/70) (both diluted at 1: 100 in FACS buffer).
   13. For unstained cells, resuspend with the same volume of FACS buffer. For microglia FMO, incubate each microglia tube with anti-CD45 or anti-CD11b. Incubate all samples at 4 °C for 20 min in the dark.
   14. Add 500 µL of FACS buffer per microtube. Centrifuge at 450 x g at 4 °C for 5 min. Discard the supernatant and resuspend the pellet with 100 µL/microtube of fixation buffer (see Table of Materials) for 20 min at room temperature in the dark.
   15. Add 500 µL/microtube of permeabilization buffer 1x (see Table of Materials) and incubate at 4 °C for 5 min in the dark.
   16. Centrifuge all the microtubes at 450 x g at 4 °C for 5 min. Discard the supernatant. Resuspend astrocytes and microglia staining tubes and also microglia FMO tubes with 50 µL/microtube of intracellular antibody anti-GFAP (APC, clone GA5) in a 1:100 dilution in 1x permeabilization buffer. For unstained cells and astrocyte FMO, resuspend the cells with the same volume of FACS buffer. Incubate all samples at 4 °C for 30 min in the dark.
   17. Wash all tubes by adding 500 µL/microtube of 1x permeabilization buffer. Centrifuge all the microtubes at 450 x g, for 5 min at 4 °C. Resuspend each microtube in 200 µL of FACS buffer. Acquire 1 x 10⁵ events/sample on the flow cytometer.
   18. For analysis, cells must be gated first on CD11b x CD45 and then GFAP histogram.
       NOTE: Unstained and FMO controls are useful to determine gates.

2. Infection and Evaluation of Infection Rates

1. Infection of glial cells with T. gondii
   1. Plate 3 x 10⁴ microglia or astrocytes with supplemented DMEM/F12 at 37 °C and 5% CO₂ for 24 h in a pretreated 96-well flat plate (see Table of Materials).
   2. Infect each cell population with tachyzoites from T. gondii RH strain expressing yellow fluorescent protein (YFP) at a multiplicity of infection (MOI) of 1:1 (parasite:cell) diluted in 200 µL/well of supplemented RPMI (3% FBS + 0.16 mM Penicillin + 0.18 mM Streptomycin + 12.5 mM HEPES + 30 mM sodium bicarbonate, pH 7.2) (see Table of Materials). Incubate at 37 °C and 5% CO₂ for 48 h.
   3. Then, discard the supernatant and fix the cells by adding 100 µL/well of 1% paraformaldehyde (PFA) diluted in PBS at 4 °C for 24 h.
   4. Replace PFA with 100 µL/well of PBS at pH 7.2.
   5. Carefully remove the supernatant and stain cells' nuclei by pipetting 50 µL/well of DAPI (5 mg/mL) diluted in PBS pH 7.2 (1:1,000) for 1 min at room temperature in the dark.
   6. Replace DAPI staining solution with 100 µL/well of PBS (pH 7.2) and analyze it by fluorescence microscopy.
2. Infection of glial cells with T. cruzi
   1. Plate 3 x 10⁴ microglia or astrocytes with supplemented DMEM/F12 at 37 °C and 5% CO₂ for 24 h in a pretreated 96-well flat plate (see Table of Materials).
      NOTE: For each time point of infection, use a different plate.
   2. Infect the glial cells with trypomastigotes from T. cruzi Y strain at a MOI of 5:1 (parasites:cell) diluted in 200 µL/well of supplemented RPMI and incubate at 37 °C and 5% CO₂ for 2 h.
      NOTE: This step is important for the cell invasion by the parasite.
   3. Remove all of the supernatant and wash the wells by adding 200 µL/well of supplemented RPMI to remove all the extracellular parasites. Remove supernatant and add 200 µL/well of fresh supplemented RPMI.
      NOTE: This step intends to remove all parasites that did not invade the adherent cells.
   4. Incubate infected cells for 2 h, 48 h and 96 h to evaluate T. cruzi replication in glial cells¹¹.
   5. After each time point, remove the supernatant and fix the cells with 100 µL/well of methanol for 15 min at room temperature and then replace methanol with 100 µL/well of PBS (pH 7.2).
   6. After fixation, prepare the cells for the immunofluorescence assay as follows.
      1. To avoid autofluorescence, treat the cells with 100 µL/well of 50 mM NH₄Cl diluted in PBS (pH 8.0) for 15 min. Wash the cells by adding 100 µL/well of PBS and immediately remove it (repeat this step 3 times).
      2. Next, permeabilize the cells by adding 100 µL/well of 0.5% Triton diluted in PBS for 15 min. Wash cells by adding 100 µL/well of PBS and immediately remove it (repeat this step 3 times).
      3. Then, incubate the cell culture with 100 µL/well of blocking solution (5% non-fat milk + 2% BSA diluted in PBS [pH 7.2]) for 1 h at room temperature. Wash cells by adding 100 µL/well of PBS and immediately remove it (repeat this step 3 times).
      4. In order to stain amastigotes, incubate with 30 µL/well of non-commercial monoclonal antibody (mAb) 2C2 anti-Ssp-4 protein (1:200) diluted in blocking solution for 1 h at room temperature.
      5. Wash cells by adding 100 µL/well of PBS and immediately remove it (repeat this step 3 times). Incubate the plate with 30 µL/well of secondary anti-mouse antibody (1:500) diluted in PBS for 1 h at room temperature.
         NOTE: The non-commercial mAb 2C2 anti-Ssp-4 protein was a kind gift from Prof. Dr. Renato A. Mortara from Department of Microbiology, Immunology and Parasitology of Federal University of São Paulo.
      6. Carefully remove the supernatant and stain nuclei by adding 50 µL/well of DAPI (5 mg/mL) diluted in PBS pH 7.2 (1:1,000) for 1 min at room temperature in the dark.
      7. Replace DAPI staining solution with 100 µL/well of PBS pH 7.2 and analyze it by fluorescence microscopy.

Results

On the 14^th 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...

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

List of materials used in this article

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 μmm 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 mm x 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 °C with trypsin