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.

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

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. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

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 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 ...

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 increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

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 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 ...

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 various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

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

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

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

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

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

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

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

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin 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. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

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 recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

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 Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

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