The study was supported by grants from the National Natural Science Foundation of China (81960226, 81760223), the Natural Science Foundation of Yunnan Province (202001AS070045, 202301AY070001-011), and the Scientific Research Foundation of Yunnan Province Department of Education (2023Y0784).

This research received approval from the Animal Experiment Ethics Committee of Kunming Medical University (kmmu20220945). All experiments adhered to established animal care guidelines. Leptomeningeal lymphatic endothelial cells (LLECs) were harvested from male C57Bl/6J mice aged 6-8 weeks and weighing between 20-25 g. These mice were procured from Kunming Medical University in Kunming, China. The entire experimental procedure was conducted under strict sterile conditions. All the centrifugation steps are performed at room temperature unless otherwise specified.
1. Preparation of reagents and instruments
NOTE: All steps involving solutions must be conducted within a class II biohazard cabinet.
<ol>
	<li>Prepare the washing buffer by mixing phosphate-buffered saline (PBS) with calcium and magnesium, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (P/S) (see Table of Materials). Keep this buffer cold at 4 &#176;C. It is essential to prepare it fresh on the day of use and degas the buffer.</li>
	<li>Prepare the digestive enzymes by combining 10 mL of PBS (without calcium and magnesium) with 2 mL of 0.25% trypsin, 1 mL of 20 mg/mL papain, and 200 &#181;L of 1 mg/mL collagenase I (see Table of Materials). 
	NOTE: Ensure the use of aliquots of appropriate volumes to prevent repeated freeze-thaw cycles. Store these aliquots at -20 &#176;C.</li>
	<li>Prepare the culture medium by utilizing the commercially available endothelial cell growth medium kit, which includes VEGF-C (100 ng/mL) and 1% P/S (see Table of Materials).</li>
	<li>Prepare the stopping buffer by supplementing Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% FBS to stop the digestion process.</li>
	<li>Prepare sterile surgical instruments: these instruments should include scissors, serrated-tip tweezers, and fine-point tweezers.</li>
</ol>
2. Flask coating
<ol>
	<li>Coat the T25 flask with a 100 &#181;g/mL fibronectin solution (see Table of Materials) and incubate overnight at 37 &#176;C. Prior to use, remove the coating solution using a pipette.</li>
	<li>Wash the flask three times with PBS, subsequently aspirate the PBS, and allow the T25 flask to air dry.</li>
</ol>
3. Leptomeninges dissociation
NOTE: Always use pre-cooled buffers and solutions at 4 &#176;C.
<ol>
	<li>Anesthetize the mice with excess inhalation of 4% isoflurane and then sacrifice them by swift decapitation using scissors that have been previously cleaned with 70% ethanol.</li>
	<li>Carefully incise the skin along the midline, starting from the opening at the back of the skull and extending toward the frontal area.</li>
	<li>Delicately remove the skull, lifting it gently with the scissors to avoid damaging the leptomeninges, ensuring the entire brain, including the leptomeninges, is obtained.</li>
	<li>Submerge the whole brain in a washing buffer and gently flush it to remove surface blood.</li>
	<li>Transfer the brain into a sterile Petri dish without chopping, and extract the leptomeninges from the brain&#39;s surface using fine-point tweezers under a microscope.</li>
	<li>Cut the leptomeninges tissue into fragments using sterile micro-scissors.</li>
</ol>
4. Leptomeninges enzymatic digestion
<ol>
	<li>Add 10 mL of the enzyme mix (step 1.2) to the fragments and incubate at 37 &#176;C for 15 min. Gently agitate to ensure the fragments detach from the bottom of the tube. Afterward, add 10 mL of stopping buffer (step 1.4) to halt the digestion.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C and carefully remove the supernatant using a pipette.</li>
	<li>Add 10 mL of cold PBS, and pass the mixture through a 70 &#181;m strainer into a sterile 50 mL tube to filter out any clumps.</li>
</ol>
5. Cell expansion
<ol>
	<li>Plate 1 x 105 cells per cm2 into a fibronectin-coated T25 flask (step 2) with 5 mL of culture medium.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h. Afterward, remove the medium to eliminate non-attached cells.</li>
	<li>Sustain the culture by replacing 50% of the medium every two days. Repeat this process two or three times.</li>
</ol>
6. Magnetic cell selection
NOTE: Work swiftly, maintain cell coldness, and utilize pre-cooled solutions to prevent non-specific cell labeling.
<ol>
	<li>Preparation of instruments and reagents: attach a magnetic separator to a magnetic separator stand (see Table of Materials). Connect a selection column to the magnetic separator, and position a 70 &#181;m cell strainer atop the selection column. Place a 50 mL tube beneath the selection column to collect the flow.</li>
	<li>Enzymatic digestion: once the cells reach 80% confluence, aspirate the medium and rinse the cells with PBS. Add 0.25% trypsin to detach adherent cells and incubate at 37 &#176;C for 5 min. Stop the digestion by adding the stopping buffer. Centrifuge the cells at 300 x g for 5 min at room temperature and remove the supernatant.</li>
	<li>Antibody incubation: resuspend 1 x 107 total cells in 100 &#181;L of PBS and add 10 &#181;L of LYVE-1 antibody (see Table of Materials). Mix thoroughly and incubate for 30 min in the dark at 4 &#176;C.
	<ol>
		<li>Spin the cells at 300 x g for 5 min and discard the supernatant. Rinse the cells by introducing 1 mL of PBS and centrifuging at 300 x g for 5 min. Completely remove the supernatant.</li>
	</ol>
	</li>
	<li>Microbeads labeling: resuspend the cells in 100 &#181;L of PBS and add 20 &#181;L of Microbeads (see Table of Materials). Mix well and incubate for 30 min in the dark at 4 &#176;C.
	<ol>
		<li>Spin the cells at 300 x g for 5 min and decant the supernatant. Subsequently, cleanse the cells with 1 mL of PBS through centrifugation at 300 x g for 5 min. Finally, completely remove the supernatant.</li>
	</ol>
	</li>
	<li>Magnetic negative exclusion: resuspend the cells in 4 mL of PBS. Pass the cells through a 70 &#181;m cell strainer to eliminate clumps. Prepare the selection column (see Table of Materials) by rinsing it with 3 mL of PBS, then apply the cell suspension into the selection column.
	<ol>
		<li>Perform washing steps with 3 mL of PBS and collect LYVE-1-negative cells into a 50 mL tube positioned below the selection column to allow them to pass through.</li>
	</ol>
	</li>
	<li>Magnetic positive selection: pipette 6 mL of PBS into the selection column. Instantly flush out the magnetically labeled cells by firmly pushing the plunger into the selection column to obtain LYVE-1-positive LLECs. 
	&#8203;NOTE: Always wait until the selection column reservoir is empty before proceeding to the next step.</li>
</ol>
7. LLECs culture
<ol>
	<li>Centrifuge the LYVE-1-positive LLECs at 300 x g for 5 min, and then carefully remove the supernatant.</li>
	<li>Plate 1 x 105 cells per cm2 into a fibronectin-coated T25 flask with 5 mL of culture medium.</li>
	<li>Maintain the culture by replacing 50% of the medium every other day. When the cells reach 80% confluence, detach the cells with 0.25% trypsin and perform cell passage. Utilize the cells for subsequent experiments after 2-3 passages.</li>
</ol>
8. Flow cytometric analysis
NOTE: The Flow cytometric analysis was conducted following the previously described procedure15.
<ol>
	<li>Add 0.25% trypsin to detach adherent cells and incubate at 37 &#176;C for 5 min. Then, add the stopping buffer to halt the digestion.</li>
	<li>Centrifuge the cells at 300 x g for 5 min and completely aspirate the supernatant. Resuspend 1 x 106 cells in 100 &#181;L of PBS.</li>
	<li>Add 1 &#181;L of LYVE-1 antibody. Mix thoroughly and incubate for 10 min in the dark at room temperature.</li>
	<li>Resuspend the cells in an appropriate amount of PBS buffer for analysis using flow cytometry and FlowJo software (see Table of Materials).</li>
</ol>
9. Immunofluorescent staining
NOTE: The immunofluorescent staining was conducted following the procedure described previously16.
<ol>
	<li>Wash the cells with PBS three times. Fix the cells with 4% paraformaldehyde (PFA) for 10 min at room temperature. Discard the PFA and wash the cells with PBS three times.</li>
	<li>Apply the block buffer for 1 h at room temperature. Remove the block buffer from the coverslips.</li>
	<li>Add the LYVE-1 antibody in the staining buffer to the cells and incubate in the dark. Wash the cells three times with PBS.</li>
	<li>Add an appropriate secondary antibody (see Table of Materials) in the staining buffer to the cells and incubate in the dark. Wash the cells three times with PBS.</li>
	<li>Counterstain with 4&#39;,6-diamidino-2-phenylindole (DAPI). The cells are now ready for immunofluorescence microscopy.</li>
</ol>

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

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The authors declare that they have no conflict of interest to disclose.

The existing protocol for harvesting and culturing LLECs in vitro has not been previously reported. This study introduces a reproducible, multi-procedural protocol for harvesting and culturing LLECs from mouse leptomeninges.
While this multi-procedural protocol is reproducible, there are several key considerations. For example, fibronectin-coated T25 flasks promote the adhesion of LLECs and function by eliminating non-adherent cells, thereby ensuring a more homogenous cellular populat...

The newly discovered leptomeningeal lymphatic endothelial cells (LLECs) form a meshwork of individual cells within the leptomeninges, exhibiting a distinct distribution pattern when compared to peripheral lymphatic endothelial cells1,2. The cellular functions and clinical implications associated with LLECs remain largely uncharted territory. In order to pave the way for functional research on LLECs, it is imperative to establish an in vitro model for their study. Therefore, this study has devised a comprehensive protocol for the isolation and primary culture of LLECs.
Mice are the preferred animal model due to their suitability for genetic manipulation in disease research. Previous studies have successfully isolated lymphatic endothelial cells from various mouse tissues, including lymph nodes3, mesenteric tissue4, dermal tissue5, collecting lymphatics6, and lung tissue7. These isolation procedures have primarily relied on techniques such as magnetic-activated cell sorting (MACS) and flow cytometry sorting8,9,10. Additionally, research efforts have led to the establishment of rat arachnoid cell lines and rat lymphatic capillary cell lines11,12. Despite the existence of explant culture techniques for leptomeninges13, there exists an urgent need for a standardized protocol for the isolation and culture of LLECs. Consequently, this study has successfully harvested and cultured LLECs by meticulously dissociating leptomeninges under the guidance of a microscope and promoting LLECs expansion through the use of vascular endothelial growth factor-C (VEGF-C). The distinctive marker for lymphatic endothelial cells is lymphatic vessel hyaluronic receptor-1 (LYVE-1)14. This multi-step protocol selectively isolates LYVE-1-positive LLECs using MACS and subsequently verifies their purity through flow cytometric analysis and immunofluorescent staining.
The primary steps of this multi-step protocol can be summarized as follows: flask coating, dissociation of leptomeninges, enzymatic digestion of leptomeninges, cell expansion, magnetic cell selection, and subsequent culture of LLECs. Finally, the purity of the isolated LLECs is confirmed through flow cytometric analysis and immunofluorescent staining. The overarching aim of this study is to present a reproducible, multi-step protocol for the isolation of LLECs from mouse leptomeninges and their subsequent in vitro culture. This protocol is poised to greatly facilitate investigations into the cellular functions and clinical implications of LLECs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Block buffer</td>
			<td>Beyotime</td>
			<td>P0102</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Solarbio</td>
			<td>C8140</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>P0131</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Solarbio</td>
			<td>11995</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Solarbio</td>
			<td>D1041</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>C-3202</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Solarbio</td>
			<td>S9010</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Solarbio</td>
			<td>F8180&#160;</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td>V10.8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE-1 antibody</td>
			<td>eBioscience</td>
			<td>12-0443-82</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>Magnetic separator</td>
			<td>Miltenyi</td>
			<td>130-042-302</td>
			<td>Sterile before use</td>
		</tr>
		<tr>
			<td>Magnetic separator stand</td>
			<td>Miltenyi</td>
			<td>130-042-303</td>
			<td>Sterile before use</td>
		</tr>
		<tr>
			<td>Microbeads</td>
			<td>Miltenyi</td>
			<td>130-048-801</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>P/S</td>
			<td>Solarbio</td>
			<td>P1400</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Solarbio</td>
			<td>G8430-25g</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>D1040</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>PDPN antibody</td>
			<td>Santa</td>
			<td>sc-53533</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>PROX1 antibody</td>
			<td>Santa</td>
			<td>sc-81983</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>Selection column&#160;</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>Sterile before use</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>VEGF-C&#160;</td>
			<td>Abcam</td>
			<td>ab51947</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>VEGFR-3 antibody</td>
			<td>Santa</td>
			<td>sc-514825</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
	</tbody>
</table>

harvest and primary culture of leptomeningeal lymphatic endothelial cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

This study presents a reproducible, multi-step protocol for harvesting lymphatic endothelial cells (LLECs) from mice and subsequently establishing their primary culture in vitro. The key steps involve flask preparation and fibronectin coating, dissociation of leptomeninges, obtaining a single-cell suspension through enzymatic digestion, and inducing LLECs expansion with VEGF-C. LYVE-1-positive LLECs are then selectively isolated using magnetic-activated cell sorting (MACS). Finally, immunofluorescence staining a...

Watch this Scientific Journal Video about Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells at JoVE.com

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

harvest-primary-culture-leptomeningeal-lymphatic-endothelial

Research

JoVE Journal

Neuroscience

1.4K Views.  The First Affiliated Hospital of Kunming Medical University. Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Video: Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...