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 2.7 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.
<ol>
	<li>Day 1
	<ol>
		<li>Prepare pure Hank&#39;s balanced salt solution (HBSS) and HBSS + 10% of heat-inactivated fetal bovine serum (FBS) (see Table of Materials).</li>
		<li>Prepare supplemented Dulbecco&#39;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 &#181;m filter (see Table of Materials). 
		NOTE: All culture media should be stored at 4 &#176;C, but it is necessary to prewarm them at 37 &#176;C for 20 min before starting the experiment.</li>
		<li>Sterilize all surgical instruments (scissors, spatula and tweezers) in an autoclave and use 70% ethanol during the procedure.</li>
		<li>Put newborns (up to 3 day-old) mice in a sealed chamber containing cotton soaked with isoflurane for 5 min for profound anesthesia.</li>
		<li>Spray the mouse pup with 70% ethanol and decapitate the animal with scissors.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Add 3 mL of trypsin per tube and place in a water bath at 37 &#176;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.</li>
		<li>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.</li>
		<li>Wash the tissue with 3 mL per tube of pure HBSS and carefully remove the supernatant. Repeat this step 2 times.</li>
		<li>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.</li>
		<li>After homogenization, centrifuge tubes at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet with 4 mL of HBSS + 10% FBS per tube.</li>
		<li>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.</li>
		<li>Place the flask in the incubator at 37 &#176;C and 5% CO2 for 30 min for adherence.</li>
		<li>Add 10 mL of supplemented DMEM/F12 per flask and incubate at 37 &#176;C and 5% CO2. 
		NOTE: The final volume is 14 mL per flask.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Day 14
	<ol>
		<li>After removing 6 mL of the supernatant and adding 7 mL of fresh supplemented DMEM/F12 medium, close the T-75 flasks tightly.</li>
		<li>Place them in the floor orbital shaker at 200 rpm and 37 &#176;C overnight to mechanically dissociate microglia from astrocytes.</li>
	</ol>
	</li>
	<li>Day 15
	<ol>
		<li>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.</li>
		<li>Next, add 4 mL of trypsin into each flask and incubate for 5 min at 37 &#176;C in order to detach the astrocytes.</li>
		<li>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.</li>
		<li>Centrifuge all tubes containing dissociated microglia and astrocytes at 450 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant. Resuspend the microglia pellet in 1 mL and the astrocytes in 10 mL of supplemented DMEM/F12.</li>
		<li>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 &#176;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.</li>
		<li>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.</li>
		<li>Add a FMO (Fluorescence Minus One)23 and an unstained sample for each cell population (astrocytes and microglia) as controls of flow cytometry assay.</li>
		<li>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.</li>
		<li>Centrifuge all microtubes at 450 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the pellet with 500 &#181;L/microtube of FACS buffer (phosphate-buffered saline (PBS) + 0.5% bovine serum albumin (BSA) + 2 mM EDTA, pH 7.2).</li>
		<li>Centrifuge all microtubes at 450 x g at 4 &#176;C for 5 min. Discard the supernatant and resuspend the pellet with 100 &#181;L/microtube of anti-CD16/CD32 (clone 93) (1:50) diluted in FACS buffer. Incubate for 10 min at room temperature.</li>
		<li>Add 500 &#181;L/microtube of FACS buffer into all microtubes and centrifuge at 450 x g at 4 &#176;C for 5 min. Discard the supernatant. Resuspend astrocytes and microglia staining tubes and also astrocyte FMO tube with 50 &#181;L/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).</li>
		<li>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 &#176;C for 20 min in the dark.</li>
		<li>Add 500 &#181;L of FACS buffer per microtube. Centrifuge at 450 x g at 4 &#176;C for 5 min. Discard the supernatant and resuspend the pellet with 100 &#181;L/microtube of fixation buffer (see Table of Materials) for 20 min at room temperature in the dark.</li>
		<li>Add 500 &#181;L/microtube of permeabilization buffer 1x (see Table of Materials) and incubate at 4 &#176;C for 5 min in the dark.</li>
		<li>Centrifuge all the microtubes at 450 x g at 4 &#176;C for 5 min. Discard the supernatant. Resuspend astrocytes and microglia staining tubes and also microglia FMO tubes with 50 &#181;L/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 &#176;C for 30 min in the dark.</li>
		<li>Wash all tubes by adding 500 &#181;L/microtube of 1x permeabilization buffer. Centrifuge all the microtubes at 450 x g, for 5 min at 4 &#176;C. Resuspend each microtube in 200 &#181;L of FACS buffer. Acquire 1 x 105 events/sample on the flow cytometer.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Infection and Evaluation of Infection Rates
<ol>
	<li>Infection of glial cells with T. gondii
	<ol>
		<li>Plate 3 x 104 microglia or astrocytes with supplemented DMEM/F12 at 37 &#176;C and 5% CO2 for 24 h in a pretreated 96-well flat plate (see Table of Materials).</li>
		<li>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 &#181;L/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 &#176;C and 5% CO2 for 48 h.</li>
		<li>Then, discard the supernatant and fix the cells by adding 100 &#181;L/well of 1% paraformaldehyde (PFA) diluted in PBS at 4 &#176;C for 24 h.</li>
		<li>Replace PFA with 100 &#181;L/well of PBS at pH 7.2.</li>
		<li>Carefully remove the supernatant and stain cells&#39; nuclei by pipetting 50 &#181;L/well of DAPI (5 mg/mL) diluted in PBS pH 7.2 (1:1,000) for 1 min at room temperature in the dark.</li>
		<li>Replace DAPI staining solution with 100 &#181;L/well of PBS (pH 7.2) and analyze it by fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Infection of glial cells with T. cruzi
	<ol>
		<li>Plate 3 x 104 microglia or astrocytes with supplemented DMEM/F12 at 37 &#176;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.</li>
		<li>Infect the glial cells with trypomastigotes from T. cruzi Y strain at a MOI of 5:1 (parasites:cell) diluted in 200 &#181;L/well of supplemented RPMI and incubate at 37 &#176;C and 5% CO2 for 2 h. 
		NOTE: This step is important for the cell invasion by the parasite.</li>
		<li>Remove all of the supernatant and wash the wells by adding 200 &#181;L/well of supplemented RPMI to remove all the extracellular parasites. Remove supernatant and add 200 &#181;L/well of fresh supplemented RPMI. 
		NOTE: This step intends to remove all parasites that did not invade the adherent cells.</li>
		<li>Incubate infected cells for 2 h, 48 h and 96 h to evaluate T. cruzi replication in glial cells11.</li>
		<li>After each time point, remove the supernatant and fix the cells with 100 &#181;L/well of methanol for 15 min at room temperature and then replace methanol with 100 &#181;L/well of PBS (pH 7.2).</li>
		<li>After fixation, prepare the cells for the immunofluorescence assay as follows.
		<ol>
			<li>To avoid autofluorescence, treat the cells with 100 &#181;L/well of 50 mM NH4Cl diluted in PBS (pH 8.0) for 15 min. Wash the cells by adding 100 &#181;L/well of PBS and immediately remove it (repeat this step 3 times).</li>
			<li>Next, permeabilize the cells by adding 100 &#181;L/well of 0.5% Triton diluted in PBS for 15 min. Wash cells by adding 100 &#181;L/well of PBS and immediately remove it (repeat this step 3 times).</li>
			<li>Then, incubate the cell culture with 100 &#181;L/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 &#181;L/well of PBS and immediately remove it (repeat this step 3 times).</li>
			<li>In order to stain amastigotes, incubate with 30 &#181;L/well of non-commercial monoclonal antibody (mAb) 2C2 anti-Ssp-4 protein (1:200) diluted in blocking solution for 1 h at room temperature.</li>
			<li>Wash cells by adding 100 &#181;L/well of PBS and immediately remove it (repeat this step 3 times). Incubate the plate with 30 &#181;L/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 S&#227;o Paulo.</li>
			<li>Carefully remove the supernatant and stain nuclei by adding 50 &#181;L/well of DAPI (5 mg/mL) diluted in PBS pH 7.2 (1:1,000) for 1 min at room temperature in the dark.</li>
			<li>Replace DAPI staining solution with 100 &#181;L/well of PBS pH 7.2 and analyze it by fluorescence microscopy.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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M., Kubo, Y., Kato, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-site+energy+supply+at+synapses+through+monocarboxylate+transporters+maintains+excitatory+synaptic+transmission.">On-site energy supply at synapses through monocarboxylate transporters maintains excitatory synaptic transmission.</a> Journal of Neuroscience. 34 (7), 2605-2617 (2014).</li><li>Buckman, L. B., Thompson, M. M., Moreno, H. N., Ellacott, K. L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+astrogliosis+in+the+mouse+hypothalamus+in+response+to+obesity.">Regional astrogliosis in the mouse hypothalamus in response to obesity.</a> Journal of Comparative Neurology. 521 (6), 1322-1333 (2013).</li><li>Vesce, S., Bezzi, P., Volterra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+active+role+of+astrocytes+in+synaptic+transmission.">The active role of astrocytes in synaptic transmission.</a> Cellular and Molecular Life Sciences. 56 (11-12), 991-1000 (1999).</li><li>Arcuri, C., Mecca, C., Bianchi, R., Giambanco, I., Donato, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pathophysiological+Role+of+Microglia+in+Dynamic+Surveillance,+Phagocytosis+and+Structural+Remodeling+of+the+Developing+CNS.">The Pathophysiological Role of Microglia in Dynamic Surveillance, Phagocytosis and Structural Remodeling of the Developing CNS.</a> Frontiers in Molecular Neuroscience. 10, 191 (2017).</li><li>Floden, A. M., Combs, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+repetitively+isolated+from+in+vitro+mixed+glial+cultures+retain+their+initial+phenotype.">Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype.</a> Journal of Neuroscience Methods. 164 (2), 218-224 (2007).</li><li>Schildge, S., Bohrer, C., Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+cortical+astrocytes.">Isolation and culture of mouse cortical astrocytes.</a> Journal of Visualized Experiments. (71), e50079 (2013).</li><li>Sarkar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+refined+CD11b+magnetic+isolation+of+primary+microglia+with+enhanced+purity+and+versatility.">Rapid and refined CD11b magnetic isolation of primary microglia with enhanced purity and versatility.</a> Journal of Visualized Experiments. (122), e55364 (2017).</li><li>Tamashiro, T. T., Dalgard, C. L., Byrnes, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+microglia+isolation+from+mixed+glial+cell+cultures+of+neonatal+rat+brain+tissue.">Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.</a> Journal of Visualized Experiments. (66), e3814 (2012).</li></ol>

The authors have nothing to disclose.

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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anti-mouse IgG (FITC)</td><td>Kirkeegood&amp;amp;Perry Lab (KPL)</td><td>Cat.: 172-1806</td><td>Immunofluorescence antidoby</td></tr><tr><td>HBSS - Hank&#039;s Balanced Salt Solution</td><td>Gibco</td><td>Cat: 14175079</td><td>Cell culture medium</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>Cat: H4034</td><td>Cell culture medium supplement</td></tr><tr><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&amp;aacute;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&amp;eacute;</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 60 mm x 15 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 &amp;deg;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 ...

concomitant-isolation-primary-astrocytes-microglia-for-protozoa

Research

JoVE Journal

Immunology and Infection

7.6K Views.  Universidade Federal de São Paulo (UNIFESP). Dit protocol beschrijft een methode die is gericht op het extraheren in parallel de twee meest voorkomende gliacel populaties, astrocyten en microglia, uit postnatale muizen. Onze methode verschilt van andere eerder beschreven protocollen voor astrocyten en microglia isolatie, omdat we ons richten op het verkrijgen en isoleren van beide celtypen in dezelfde extractie in dezelfde voorwaarde, waardoor het gebruik van experimentele dieren wordt geoptimaliseerd en de studie van relatieve rollen v...

Video: Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

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 cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

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

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 for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

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

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Neuroscience

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

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