Name: concomitant isolation of primary astrocytes and microglia for protozoa parasite infection
Uploaded: 2020-03-18T15:00:00
Description: Watch this Scientific Journal Video about Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection at JoVE.com

We would like to thank professor Dr. Renato A. Mortara from Federal University of S&#227;o Paulo (UNIFESP) for mAb 2C2 anti-Ssp-4. This work was supported by grants from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP, grant 2017/25942-0 to K.R.B.), Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq, grant 402100/2016-6 to K.R.B.), Instituto Nacional de Ci&#234;ncia e Tecnologia de Vacinas (INCTV/CNPq) and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel 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.

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 S&#227;o 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....

<ol>
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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 en...

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.

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	<tbody>
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			<td>70% Ethanol</td>
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			<td>Anti-GFAP antibody</td>
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			<td>Immunofluorescence antidoby</td>
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			<td>Culture medium filter</td>
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			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
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			<td>FACS Buffer preparation</td>
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			<td>CD11b (FITC)</td>
			<td>BD Pharmigen</td>
			<td>Cat.: 553310</td>
			<td>Flow cytometry antibody</td>
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			<td>Invitrogen</td>
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			<td>Flow cytometry antibody</td>
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			<td>Centrifuge</td>
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			<td>Centrifuge</td>
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			<td>5415R</td>
			<td>Centrifugation</td>
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			<td>Pachane</td>
			<td>Cat: 200</td>
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			<td>CO2 Incubator</td>
			<td>ThermoScientific</td>
			<td>Model: 3110</td>
			<td>Primary cells maintenance</td>
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			<td>Conical tubes 15 mL</td>
			<td>Corning</td>
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			<td>Conical tubes 50 mL</td>
			<td>Corning</td>
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			<td>Plastic material</td>
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			<td>Countess automated cell counter</td>
			<td>Invitrogen</td>
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			<td>Cell counter</td>
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			<td>DAPI</td>
			<td>Invitrogen</td>
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			<td>Immunofluorescence antidoby</td>
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			<td>Nikon</td>
			<td>Cat: DS-RI1</td>
			<td>Capture images on microscope</td>
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			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>Cat: 12800-058</td>
			<td>Cell culture medium</td>
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			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>Cat: E9884</td>
			<td>FACS Buffer preparation</td>
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			<td>F12 Nutrient Mixture</td>
			<td>Gibco</td>
			<td>Cat: 21700-026</td>
			<td>Cell culture medium</td>
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			<td>FACS Canto II</td>
			<td>BD Biosciences</td>
			<td>Unavaiable</td>
			<td>Flow cytometer</td>
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			<td>Fetal Bovine Serum (FBS)</td>
			<td>LGC Biotechnology</td>
			<td>Cat: 10-bio500-1</td>
			<td>Cell culture medium supplement</td>
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			<td>Flow Jo (software)</td>
			<td>Flow Jo</td>
			<td>Version: Flow Jo_9.9.4</td>
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			<td>Fluorescence intenselight</td>
			<td>Nikon</td>
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			<td>GFAP (APC)</td>
			<td>Invitrogen</td>
			<td>Cat.: 50-9892-82</td>
			<td>Flow cytometry antibody</td>
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			<td>Goat - anti-mouse IgG (FITC)</td>
			<td>Kirkeegood&#38;Perry Lab (KPL)</td>
			<td>Cat.: 172-1806</td>
			<td>Immunofluorescence antidoby</td>
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			<td>HBSS - Hank&#39;s Balanced Salt Solution</td>
			<td>Gibco</td>
			<td>Cat: 14175079</td>
			<td>Cell culture medium</td>
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			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>Cat: H4034</td>
			<td>Cell culture medium supplement</td>
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			<td>IC Fixation Buffer</td>
			<td>Invitrogen</td>
			<td>Cat: 00-8222-49</td>
			<td>Cell fixation for Flow Citometry</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Model: ECLIPSE TS100</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Crist&#225;lia</td>
			<td>Cat: 21.2665</td>
			<td>Inhaled anesthetic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Synth</td>
			<td>Cat: 01A1085.01.BJ</td>
			<td>Fixation for Immunofluorescence</td>
		</tr>
		<tr>
			<td>Micro spatula</td>
			<td>ABC stainless</td>
			<td>Unavaiable</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Microtube 1.5 mL</td>
			<td>Axygen</td>
			<td>Cat: MCT-150-C</td>
			<td>Plastic material</td>
		</tr>
		<tr>
			<td>Monoclonal antibody (mAb) 2C2 anti-Ssp-4</td>
			<td>Non commercial</td>
			<td>Non commercial</td>
			<td>Immunofluorescence antidoby</td>
		</tr>
		<tr>
			<td>Multichannel Pipette (p200)</td>
			<td>Corning</td>
			<td>Cat: 751630124</td>
			<td>Pipette reagents</td>
		</tr>
		<tr>
			<td>NIS Elements Software</td>
			<td>Nikon</td>
			<td>Version 4.0</td>
			<td>Acquire and analyse images</td>
		</tr>
		<tr>
			<td>Non-fat milk</td>
			<td>Nestl&#233;</td>
			<td>Cat: 9442405</td>
			<td>Blocking solution for immunofluorescence</td>
		</tr>
		<tr>
			<td>Orbital Shaker Incubator</td>
			<td>ThermoScientific</td>
			<td>Model: 481 Cat: 11</td>
			<td>Dissociate microglia from astrocytes</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>Cat: P6148</td>
			<td>Fixation for Immunofluorescence</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Non commercial</td>
			<td>Non commercial</td>
			<td>Neutral Buffer</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma Aldrich</td>
			<td>Cat: P-7794</td>
			<td>Cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer (10X)</td>
			<td>Invitrogen</td>
			<td>Cat: 00-8333-56</td>
			<td>Cell permeabilization for Flow Citometry</td>
		</tr>
		<tr>
			<td>Petri dish 60x15 mm (Disposable, sterile)</td>
			<td>Prolab</td>
			<td>Cat: 0303-8</td>
			<td>Plastic material</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Kasvi</td>
			<td>K39-1014B</td>
			<td>Calibrate pH solution</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>Cat: 31800-014</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>ABC stainless</td>
			<td>Cat: LO9-W4</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			<td>Corning</td>
			<td>Cat: 4101</td>
			<td>Plastic material</td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			<td>Corning</td>
			<td>Cat: 4051</td>
			<td>Plastic material</td>
		</tr>
		<tr>
			<td>Single Channel Pipette (p1000)</td>
			<td>Gilson Pipetman</td>
			<td>Cat: F123602</td>
			<td>Pipette reagents</td>
		</tr>
		<tr>
			<td>Single Channel Pipette (p200)</td>
			<td>Gilson Pipetman</td>
			<td>Cat: F123601</td>
			<td>Pipette reagents</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>Cat: S6297</td>
			<td>Cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Streptomycin sulfate salt</td>
			<td>Sigma Aldrich</td>
			<td>Cat: S9137</td>
			<td>Cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>Cat: T9284</td>
			<td>Permeabilization for immunofluorescence</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>Cat: 27250-018</td>
			<td>Digestive enzyme</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>ABC stainless</td>
			<td>Cat: L28-P4-172</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Novatecnica</td>
			<td>Model: 09020095</td>
			<td>Digeste tissue at 37 &#186;C with trypsin</td>
		</tr>
	</tbody>
</table>

concomitant isolation of primary astrocytes and microglia for protozoa parasite infection

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.

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...

Watch this Scientific Journal Video about Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection at JoVE.com

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

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 ...

This protocol describes a method that is focused on extracting in parallel the two most abundant glial cell populations, astrocytes and microglia, from postnatal mice. Our method differs from others previously described protocols for astrocyte and microglia isolation because we focus on obtaining and isolating both cell types in the same extraction at the same condition, thus optimizing the use of experimental animals and improving the study of relative roles for these cells in immunological contexts. This protocol can be useful for better investigating microglia and astrocyte roles in many contexts and diseases in the central nervous system such as Alzheimer, Parkinson, and infections by for example protozoa parasites or viruses.Obtaining and isolating both cell types from the same extraction is desirable to evaluate the comparative role of each cell subset immunological contexts such as infection or inflammation. People should be careful especially during brain removal and nervous tissue washes, but I believe that with this protocol and the video shot, people will not struggle. This protocol can be long depending on the number of animals to extract the cortices so be prepared to spend about four hours or more.On day one, prepare pure HBSS and HBSS plus 10%heat-inactivated FBS. Prepare supplemented DMEM F12 and filter it with a 0.22 micron filter. Sterilize all surgical instruments in an autoclave and use 70%ethanol during the procedure.Put newborn mice in a sealed chamber containing cotton soaked with isoflurane for five minutes for profound anesthesia. Spray the mouse pup with 70%ethanol and decapitate the animal with scissors. Make a sagittal cut with scissors along the cranium to open it and expose the brain.Remove the brain using a micro spatula to maintain the brain integrity. Place the brain in a dry six centimeter diameter Petri dish. Using a micro spatula, remove the olfactory bulb and cerebellum.Move the cortex to another Petri dish containing two milliliters of HBSS plus 10%FBS. Cut the brain tissue into small pieces with sterile scissors and using a P1000 micropipette, transfer each brain tissue with HBSS plus 10%FBS to different 15 milliliter conical tubes. If necessary, use HBSS plus 10%FBS to fill the final volume to two milliliters.Wash the cut brain tissue with three milliliters of HBSS plus 10%FBS for each tube. After decantation, carefully remove the supernatant. Repeat the wash with HBSS plus 10%FBS three additional times.Then wash with three milliliters of pure HBSS three times. After decantation, carefully remove the supernatant. Next, add three milliliters of trypsin and place the tube in a water bath at 37 degrees Celsius for 30 minutes gently shaking the tubes every five minutes.This digests the collected tissue. Avoid bubbles. To inactivate the trypsin, wash the tissue with three milliliters of HBSS plus 10%FBS and after decantation of the tissue, carefully remove the supernatant.Repeat this wash three times. Then wash the tissue with three milliliters of pure HBSS and repeat two additional times. After decantation of the tissue, carefully remove the supernatant.Add seven milliliters of pure HBSS and homogenize the tissue through successive passages in pipettes, first with a 10 milliliter serological pipette, followed by a five milliliter pipette, and finally with a P1000 micropipette. After homogenization, centrifuge the tubes at 450 times g for five minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet with four milliliters of HBSS plus 10%FBS for each tube.Transfer the cells to a commercially pretreated T75 flask for optimal cell culture adhesion. Place the flask in an incubator at 37 degrees Celsius at 5%carbon dioxide for 30 minutes for adherence. Then add 10 milliliters of supplemented DMEM F12 to the flask and incubate at 37 degrees Celsius and 5%carbon dioxide for two nights.On day three, remove seven milliliters of culture medium from the T75 flask and add seven milliliters of fresh supplemented DMEM F12. Return the flask to the incubator. On day five, to remove debris and nonadherent cells, exchange all medium from the T75 flask with 14 milliliters of fresh supplemented DMEM F12.Then every 48 hours, remove six milliliters of the supernatant and add seven milliliters of fresh supplemented DMEM F12 medium until day 14 of culture. On day 14, after removing six milliliters of supernatant and adding seven milliliters of fresh supplemented DMEM F12 medium, close the T75 flasks tightly with plastic wrapping. Place them in the floor orbital shaker at 200 RPM and 37 degrees Celsius overnight to mechanically dissociate microglia from astrocytes.On day 15, 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 and transfer it to a 50 milliliter conical tube. Next, add four milliliters of trypsin to each flask and incubate for five minutes at 37 degrees Celsius in order to detach the astrocytes.Then add five milliliters of supplemented DMEM F12 to inactivate the trypsin. Wash the flasks with their own medium and transfer the contents to a 50 milliliter conical tube. Centrifuge all tubes containing dissociated microglia or astrocytes at 450 times g for five minutes at four degrees Celsius.Discard the supernatant. Resuspend the microglia pellet in one milliliter of supplemented DMEM F12 and the astrocytes in 10 milliliters of supplemented DMEM F12. 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 a pretreated flat bottom cell culture plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours to allow them to attach. On the 14th day, glial cell culture underwent mechanical dissociation.It was observed with a purity of 89.5%for the astrocyte population and 96.6%for the microglia population. Astrocytes and microglia from C57BL6 mice were infected with T.cruzi Y.After two hours, 48 hours, and 96 hours, chambers were fixed with methanol and stained with cell nuclease marker DAPI and anti-GFAP. The use of genetically modified parasites expressing fluorescent reporters or parasites labeled with specific fluorescent antibodies improved the immunofluorescence microscopy since they are better distinguished from the cell nucleus.Two examples are presented here. T.gondii RH strain constitutively expressing YFP and T.cruzi Y strain stained with non-commercial monoclonal antibody 2C2 anti-SSP4 protein. It is important to carefully wash the cells to not lose any cell aggregates.After glial cells infection, other parameters can be evaluated such as ELISA for cytokine production present in the supernatant, Western blotting for protein expression, and real time quantitative PCR for RNA transcription evaluation. Many questions related to the function of glial cells, for example their metabolism, immune response, and cell biology itself can be addressed and benefited by this technique. All protozoa parasites are able to infect human cells as well as culture mouse cells so it is important to be careful when handling this parasite.

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...