We would like to thank members of the Tsirka lab for their advice and helpful comments. This work was supported by R01NS42168 to SET, 12PRE12060489 to RB, an NSF-3MT IGERT and a Turner Dissertation fellowship to LT, and NSF-3MT IGERT to JCN.

1. Dissection (Day 0)
<ol>
	<li>Induce anesthesia to p0-2 mouse pups with hypothermia. Wipe the heads of the pups with a Kimwipe soaked in 70% ethanol to disinfect the tissue.</li>
	<li>Remove the heads with a pair of scissors, using 4 pups per 10 cm culture plate. Place the heads in a petri dish containing ice-cold Hank's buffer.</li>
	<li>Anchor the heads using curved forceps through the eye sockets and carefully remove the skin covering the skull with straight microforceps.</li>
	<li>Remove the cranial bones with straight microforceps, using caution to not puncture or damage the cortices. The most effective way is to begin the removal starting near the cerebellum, which is located at the base of the head, as this region will be discarded.</li>
	<li>Remove the brain with a small spatula, and place the (4) brains in a fresh petri dish containing ice-cold Hank's buffer.</li>
	<li>All steps from this point on utilize microforceps and are performed under a dissection microscope. Starting on the ventral side of the brain, anchor the tissue by holding the cerebellum and make two small incisions on either side of the midbrain. Be careful not to cut all the way through the tissue, as the cortices cover the midbrain in this region.</li>
	<li>Gently tease the midbrain and cerebellum in one piece from the two cortices. The two cortices should form a concave shape.</li>
	<li>Separate the two cortices and orient one single cortex with the medial side up for further dissection. The hippocampus can be difficult to observe, but it is located opposite of the olfactory bulb which appears as a small nodule on the pointed end of the cortex.</li>
	<li>Remove the hippocampus, which has a crescent-shape. Flip the cortex over to view the dorsal side.</li>
	<li>Using the olfactory bulb as a starting point, remove all meninges and the olfactory bulb itself from the cortex.</li>
	<li>The dissected cortices should be placed into 15 ml conical tubes with 14 ml of cold Hank's buffered saline on ice.</li>
</ol>
2. Cell Culture (Day 0)
<ol>
	<li>Pre-coat 10 cm tissue culture dishes with 5 &mu;g/ml of Poly-D-Lysine (PDL) diluted in autoclaved water for 3 hr at 37 &deg;C.</li>
	<li>Aspirate PDL, and wash plates once with autoclaved water. Dry plate under the UV in the tissue culture hood for 20 min. This step should be performed just prior to the dissection process.</li>
	<li>Aspirate Hank's buffer from tubes containing the cortices from 4 brains each, apply 4 ml of 1x trypsin/EDTA solution, triturate tissue with p1000 tip, and place tubes in 37 &deg;C incubator for 15 min.</li>
	<li>Add 4 ml of complete microglial media per tube to stop the enzymatic digestion, mix, and spin down contents at 1.5K rpm for 5 min.</li>
	<li>Aspirate supernatant and repeat wash with 4 ml of complete media. Resuspend cells in 10 ml of complete microglial media (DMEM with 10% FBS, 1% sodium pyruvate, 0.08% gentamycin), and filter through a 40 micron mesh cell strainer.</li>
	<li>Plate at a density of 8 cortices per 10 ml in a 10 cm tissue culture plate and place in a tissue culture incubator at 37 &deg;C and 5% CO2 (See Adult Microglia protocol).</li>
</ol>
(Day 3)
<ol>
	<li>Change media in all cell culture dishes with complete microglial media.</li>
</ol>
(Day 10)
<ol>
	<li>Add 400 microliters of 60 mM Lidocaine in HBSS (to detach microglia) into medium of 10-cm tissue culture plates and incubate at room temperature (RT) for 10-15 min.</li>
	<li>Collect the media/cell suspension from the plate, and wash the plate once with Hank's buffer. Collect the wash buffer to recover remaining microglia.</li>
	<li>Add 5 mM EDTA (pH 8.0) to the cell suspension to a final concentration of 50 &mu;M or at a 1/100 dilution.</li>
	<li>Spin down cell suspension at 1,000 x g (1,500 rpm) for 5 min and re-suspend in 1 ml DMEM with 1% FBS.</li>
	<li>Count viable cell number (as per Adult Microglia 3.1/3.2) and split cells into tissue culture plates at the desired experimental density. Approximately 1x106 microglia are harvested from a 10 cm plate containing the cortices from 4 brains. For immunofluorescence, a density of 2.5x104 cells per 18 mm coverslip is recommended.</li>
	<li>Allow at least 24 hr for microglial cells to fully return to their ramified, resting state prior to use.</li>
</ol>
Protocol Text: (Adult Microglia)
1. Tissue Collection
<ol>
	<li>Coat tissue culture plates with Poly-D-Lysine (PDL) for 3 hr at 37 &deg;C or overnight at 4 &deg;C. Wash the plates once with autoclaved water immediately prior to use.</li>
	<li>Euthanize mouse by CO2 asphyxiation and ascertain that euthanasia is complete. Clean spinal cord area with 70% ethanol.</li>
	<li>Using a pair of scissors cut the skin on top of the spinal cord; then proceed to cut the spinal cord out from region T1 to T12. Cut the remaining muscle off of the sides of the spinal cord.</li>
	<li>Slowly cut the vertebrae using microscissors as you gently hold the spinal cord with your fingertips. Be careful not to puncture the spinal cord as the tissue is very soft. Slowly extract the spinal cord using microforceps.</li>
	<li>Submerge the spinal cord in a petri dish containing ice-cold HBSS. Using a light microscope remove all visible meninges using microforceps</li>
	<li>Cut the spinal cord into transversal segments small enough to generate several fragments. The thickness of the fragments should not be more than 2 mm in order to facilitate efficient digestion. Transfer the spinal cord pieces to a 15 ml conical tube containing 1 ml ice-cold HBSS. Keep the tube on ice until the digestion step.</li>
</ol>
2. Digestion of Tissue
<ol>
	<li>Aspirate HBSS from 15 ml conical tube containing spinal cord tissue. Be careful not to aspirate any piece of tissue.</li>
	<li>Add 1 ml of trypsin (2.5 g/L irradiated porcine trypsin and 0.2 g/L EDTA in HBSS) and incubate at 37 &deg;C for 30 min.</li>
	<li>To stop the digestion remove the trypsin and add 3 ml of primary microglia medium which contains serum to halt the enzymatic digestion.</li>
	<li>Pipet up and down to dislodge the tissue. Filter the cell suspension using a 40 &mu;m cell strainer.</li>
</ol>
3. Cell Counting and Plating
<ol>
	<li>Mix equal volume of cell suspension and DAPI solution.</li>
	<li>Count cells using a hemocytometer.</li>
	<li>Plate at a density between 5-7x105 cells/ml in PDL-coated 35 x 10 mm dishes . Plating at a lower density prevented cell growth even when cells were cultured in 12 well plates which have a smaller area compared to 35 x 10 mm dishes.</li>
</ol>

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Neurosci. 26, 1880-1887 (2006).</li><li>Sasmono, R., Oceandy, D., Pollard, J., Tong, W., Pavli, P., Wainwright, B., Ostrowski, M., Himes, S., Hume, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101, 1155-1163 (2003).</li><li>Ekdahl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+-+tuning+and+pruning+adult+neurogenesis.">Microglial activation - tuning and pruning adult neurogenesis.</a> Front Pharmacol. 3, 14 (2012).</li><li>Sierra, A., Gottfried-Blackmore, A., McEwen, B., Bulloch, 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+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55, 412-424 (2007).</li></ol>

Authors have nothing to disclose.

Microglia modulate CNS normal functioning as well as inflammatory responses to various pathologies. Functional synaptic remodeling by microglia has been implicated in the maintenance of normal brain homeostasis 15. During the neurogenic cascade they participate in the clearance of neural progenitor cells from the dentate gyrus of the hippocampus 4, 16. Therefore, it is necessary to develop a culture system in which to study neonatal and adult microglia, which will cover the vast swath of development and adulthood, throughout which microglia have multiple discrete functions. We have outlined a quick and efficient method to culture microglia from the adult spinal cord and neonatal brain. We were able to obtain cell preparations in the adult that were 70% microglia with the remaining percentage consisting of astrocytes, and the purity of the neonatal microglial preps approaching 100%.
	Given the role that astrocytes have in regulating the activation state of microglia 17-19, it is important to optimize the culture conditions to improve the purity of the culture. Although combining the cell suspension with an equal volume of DAPI Fluoromount allows for an accurate cell count, it is not possible to distinguish microglia from other cell types using this method. To improve the purity of the adult microglial preparation, GFP- coated Dynabeads can be incubated with the cell suspension derived from MacGreen mice, whose myeloid cells already express GFP 20. Another suggestion would be to plate the mixed cell suspension in culture dishes that have not been previously coated with PDL to prevent astrocytic attachment 13. However, we have found that no cells grow on the plate unless they have been previously coated, which in our hands is applicable to both neonatal and adult cells.
	Many protocols describing methods for isolation and plating of primary microglia from either rat or mouse brain tissue utilize shaking of mixed glial cultures to detach the microglia and thereby obtain a purified population for downstream experiments 12. Our protocol differs most noticeably in excluding the shaking step, and instead focusing on the purification of a microglial-only population through precisely titrated culturing conditions. In our experience, shaking mixed glial cultures in order to remove the microglia is effective, but has one significant drawback - the fact that the shaken microglia take many days to return to a complete resting morphology 12. This may be problematic given that activated microglia have a considerably different cytokine profile 21. Our protocol, while taking slightly longer to obtain pure microglia, maintains the cells in a resting state throughout. As with the adult prep discussed in the next paragraph, the one drawback of our method is a possibility of small contamination of other cell types, namely astrocytes.
	Our technique for culturing adult microglia varies considerably from preexisting methods. Many protocols call for the use of density gradient centrifugation (Percoll) as well as the use of flow cytometry to obtain a pure microglial population 22. In our method of creating a stable microglial culture from adult spinal cord we do not utilize either technique, which makes for a considerably less time consuming and complicated protocol for the researcher to follow. While the vast majority of adult microglial preparations come from brain tissue, the spinal cord is comprised of the same cell types - and therefore our protocol presents a fair comparison to previously described methods 22. One tradeoff in our protocol is a slightly less pure microglial preparation, consisting of 70% microglia, with the remaining fraction of cells being mostly of astrocytic origin. This discrepancy can potentially be overcome using an additional step of isolation of more pure microglia using very specific cell type markers of the microglial lineage such as Iba-1 and F4/80 conjugated to magnetic beads.
	Most studies that assess the role of microglia in neurological diseases that affect the spinal cord have been conducted using cells from the neonatal brain, and while this is a solid approach for answering questions about a role for microglia in normal development as well as pathologies affecting the brain specifically, we felt that a more specific method had to be established to study microglia function in the adult spinal cord. Our neonatal method is a very efficient way to answer most questions regarding the normal functioning of the brain as well as in certain pathological conditions, and the adult method is a good alternative to the use of neonatal cells for such studies given the variability in immunological responses between neonatal microglia and adult cells and between microglia from the brain and the spinal cord 6-9. Microglia obtained from both methods described are also appropriate for in vitro co-culture studies (we primarily culture them with neurons or oligodendrocytes in our lab) that address the effect of microglia on other cell types as well as the neonatal and adult microglial response to activating stimuli.

Microglia are the resident immune cells of the CNS, most closely resembling peripheral macrophages in structure and function 1. It has recently been demonstrated that postnatal microglial cells derive from primitive myeloid progenitors and are generated before the eighth day of embryogenesis, upending the previous notion that postnatal hematopoietic progenitors are the source of microglia in the adult brain 2. They play key roles in several neurological diseases and can quickly respond to infection or injury by releasing pro-inflammatory or anti-inflammatory cytokines 3. Thus microglia encompass a standalone unit in the CNS that can be manipulated to affect disease progression. Developing robust and reproducible methods to isolate and culture neonatal or adult microglial cells is important to future studies.
Microglia are known to be critical players in a number of brain pathologies. More recently, roles are emerging for the cells in normal brain development and function as microglia phagocytose excess neural progenitor cells from the dentate gyrus of the hippocampus 1, 4. Microglia can also modulate several neurological conditions that affect the spinal cord, such as MS, neuropathic pain, and spinal cord injury 5-7. Spinal cord microglia react differently compared to brain microglia in response to activation signals 8, 9, probably due to differences in the local environment. Thus it is important to establish an appropriate in vitro system to culture and study spinal cord microglia. Neonatal microglia produce significantly more of the pro-inflammatory cytokine nitric oxide compared to adult cells after in vitro stimulation with IFN-&gamma; or TNF-a10,11 further highlighting the need to use adult cells to study microglia in the context of certain diseases.
The protocol we employ in the lab to culture neonatal microglia is a variation of recent methods which utilize shaking of mixed glial cultures in an effort to remove the microglia from the surface of the cell culture flask 12. We also describe a method to culture microglia from the adult mouse spinal cord based on a protocol first described by Yip, et al13. This method provides a quicker way to culture adult cells compared to other available protocols 14. The resulting preparation is 70% microglia; the remaining percentage is composed of astrocytes. Although the purity of our culture is lower compared to other published methods 13, this culture system is useful for exploring the microglial in culture response to various activating stimuli as well as for the study of diseases that mainly affect the spinal cord and in which a strong inflammatory response is a main feature.
All the protocols described have been approved by the Stony Brook University IACUC.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

An example of resting and activated microglial cells is shown in Figure 1. The microglia were visualized 24 hr after plating (Figure 1a) and exhibit ramified (resting) morphology. Exposure to the priming reagent, bacterial lipopolysaccharide (LPS) results in changes in microglial morphology as the cells become activated (Figure 1b).
An example of counting the cells for plating is shown in Figure 2. Due to the presence of cell debris, DAPI Fluoromount (instead of Trypan Blue) was added to an equal volume of the cell suspension and placed in a hemocytometer for cell counting as described in 4, 13. DAPI positive cells were visualized using a fluorescent microscope. Use of the bright field setting facilitated an accurate count of the cells present (Figure 2). Around 7x105 cells were obtained per mouse spinal cord.
The cells were plated in PDL-coated 35 x 10 mm tissue culture dishes. We found that coating with PDL was a critical step, as cells did not adhere/grow unless the plates were coated. Microglia fully attach to the culture plate after approximately two days in culture (Figure 3). The spinal cord of MacGreen mice, which express GFP under the control of the microglia/macrophage promoter CSF-1R, was used for this experiment.
<img alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/50647/50647fig1highres.jpg" src="/files/ftp_upload/50647/50647fig1.jpg" /> 
 Figure 1. Representative image of neonatal microglia after two days in culture. Microglia were cultured from p0 C57/BL6 mouse pups, and plated on uncoated coverslips at a density of 2.5 x 105 cells/ml. a, c. Untreated microglia that have returned to a resting, ramified state; b, d. Microglia treated with 100 ng/ml LPS for 4 hr and show an activated, amoeboid shape; Iba1 (green, a marker specific for a macrophages/microglia cell surface protein) has been used to visualize the cells, while bright field images were used to show overall cell populations. DAPI Fluoromount was used to visualize nuclei and indicate the purity of the culture.
<img alt="Figure 2" fo:content-width="4.75in" fo:src="/files/ftp_upload/50647/50647fig2highres.jpg" src="/files/ftp_upload/50647/50647fig2.jpg" /> 
 Figure 2. Cell counting. DAPI Fluoromount was mixed with an equal volume of cell suspension and placed in a hemocytometer. We combined the brightfield and the fluorescence setting to visualize DAPI positive cells and the hemocytometer grid for an accurate cell count.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50647/50647fig3highres.jpg" src="/files/ftp_upload/50647/50647fig3.jpg" /> 
 Figure 3. Representative images of adult spinal cord microglia after two days in culture. a. The spinal cord of a MacGreen mouse was used for the culture procedure. Cells were plated in a PDL-coated 35 x 10 mm tissue culture dish at a density of 7x105 cells/ml. b. Bright field image of spinal cord microglia after two days in culture. Microglia (blue arrows) and astrocytes (red arrows) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50647/50647fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

27.8K Views.  Stony Brook University. We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Video: Culturing Microglia from the Neonatal and Adult Central Nervous System

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

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 ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...