This work was supported by the &#34;Else Kr&#246;ner-Fresenius-Stiftung&#34; (2018_A03 to TR), &#34;Innovative Medizinische Forschung (IMF) M&#252;nster&#34; (I-RU211811 to TR) and German Research Foundation (DFG, INST 2105/27-1, ME 3283/5-1, and ME 3283/6-1 to SGM). Illustrated images provided by Heike Blum.

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The authors declare no competing financial interests.

Microvascular endothelial cells provide barrier functions in all tissues and their dysfunction results in disease of the associated organs 3. Moreover, organ-specific studies of microvascular EC could pave the way for new therapeutic strategies. Therefore, a deeper understanding of microvascular EC function under physiological and pathophysiological conditions is of great scientific interest. Modulation of leukocyte/endothelium interaction is successfully used to treat multiple sclerosis patients with natalizumab, an antibody which prevents lymphocyte adhesion and thereby transmigration 6. However, various myopathies including the inflammatory subtypes still remain only poorly treatable to date. Therefore, elucidation of EC properties specifically in skeletal muscle endothelium could help to expand available treatments to myopathy or result in novel therapeutic approaches.
Immortalized endothelial cell lines (IECL) have been established and used for several in vitro models. These cell lines offer some advantages compared to primary microvascular EC due to immortalization and fast proliferation. Furthermore, primary cultured cells can undergo senescence and lose or change their morphology or physiological function after some time or passages. However, IECL only maintain some endothelial cell properties and cannot resemble the variety of different organ-borne primary microvascular EC. Of note, they are rarely derived from skeletal muscles. To date, only a single publication describes a human skeletal microvascular endothelial cell line named TSM15 5. In contrast, murine microvascular EC have not been described so far. Finally, primary cells from transgenic animals provide the opportunity to study genetic modifications in vitro. However, primary cell cultures also hold certain pitfalls. First, contamination with cells of no interest can interfere with the validity of experimental findings. Therefore, the accuracy of several selection steps and a high purity of isolated cells must be guaranteed. Cell suspension after dissociation of muscle tissue contains erythrocytes and different mononuclear cell types such as immune cells, satellite cells, pericytes, fibroblasts and endothelial cells 7. Therefore, cell fractions after the CD31 MCS step were tested for markers exclusively expressed by muscle satellite cells. As expected CD45- CD31- fraction and primary muscle cells showed Pax-7 and Cdh15 expression, whereas CD45- CD31+ and cultivated primary murine MMEC were negative for these markers after a second CD31 MCS step. Furthermore a quality control of isolated or cultured cells is inevitably to guarantee reliable and reproducible experiments. Several methods for quality control of primary murine MMEC are available: besides microscopy of morphological properties, flow cytometry, PCR and immunofluorescence stainings for characteristic EC markers (e.g. CD31, Sca-1) can be used. Purity assessment of freshly isolated primary murine MMEC was only about 60-70%, whereas a viability of about 70% could be detected. Microscopy of these cells partially shows cell conglomerates, which might consist of contaminating cells such as fibroblasts or pericytes attached to EC. After 7 days mainly flat and elongated cells and spheroid cells can be observed. However, two CD31 MCS passages resulted in high viability and purity. Alternative approaches to increase purity such as puromycin-containing media failed in primary murine MMEC while being effective in murine brain microvascular EC 8.
Since microvascular EC derived from capillaries express tight junction proteins, gene expression of Cld5, Ocln and Tjp1 in primary murine MMEC compared to differentiated muscle cells was examined. As expected all tight junction molecules were detected in primary murine MMEC. In comparison, muscle cells demonstrated only low expression of Tjp1, whereas no expression of Cld5 and Ocln could be determined. Similar expression patterns were previously demonstrated in whole murine skeletal muscle. Here, an expression on gene and protein level could be detected for Tjp1 but not for occludin 9. Further functional features such as transendothelial resistance could be measured.
The herein described protocol features only the isolation of microvascular EC from murine muscle tissue. However, it might be transferred to various other species as for example a similar protocol was published for rat microvascular EC derived from epididymal fat pads. Here, CD31 positive selection via MCS was preceded by density gradient centrifugation 10. Similar approaches were successful in macrovascular EC isolation from rat femoral arteries 11. A recently published method to isolate microvascular EC of heart and lung tissue uses CD31 and endoglin (CD105) as antigens for magnetic cell sorting 12. However, the purity of EC could not further be increased by using additional CD105 magnetic bead separation. Another possibility to isolate and purify primary murine MMEC is multicolor fluorescence activated cell sorting (FACS). A distinct population of Sca-1+, CD31+, CD34dim and CD45- cells from murine muscles could be isolated and was characterized as primary murine MMEC 13. An advantage of this method is a pure EC population which can be directly used or cultured for further experiments (Note that in our experience FACS leads to increased cell death due to higher cell stress). Further, dead cells or conglomerates of different cell types can significantly reduce the isolated cell numbers by FACS. Additionally, it is reported that primary murine MMEC isolated by FACS were unsuccessfully cultured at commonly used culture conditions of 21% O2 and 5% CO2. Here, the oxygen level had to be adjusted to 5% for a sufficient cultivation 13. Moreover, the FACS technique is more complex, time-consuming and the infrastructural prerequisites are expensive.
The musculus quadriceps femoris and the musculus triceps surae of male mice in the age of 4-12 weeks are most suitable for isolation of primary murine MEC as they are easily accessible and large enough for isolation of sufficient cell numbers (5 - 10 x 105 per gram muscle). Therefore, it must be ensured that conditions are comparable in independent experiments. As described above, primary cells can undergo senescence. Hence, use cells for respective experiments promptly in lower passages. After passage 8&#8211;10 primary murine MMEC change their morphology, demonstrate slow proliferation rates and lose their contact inhibition as signs for dedifferentiation and senescence.
Further there are some notes for troubleshooting for this protocol. Sterile working is essential to avoid contaminations. Quality controls are necessary to monitor purity and viability of isolated cells. Used materials in this protocol must be stored and applied according to manufacturer&#39;s instructions. Additional, age and sex of used animals, as well as different muscle groups might influence the quality and comparability of isolated cells in experiments.
The described protocol should be considered as platform method to isolate primary microvascular EC of skeletal muscles. Isolated cells can be used for several applications to gain further insights into blood-muscle-barrier function. Cells are suitable for both protein and RNA expression studies as well as functional assays (including transmigration and adhesion studies). However, this protocol was optimized for studies focusing on inflammatory processes and hence slight modifications might be necessary with respect to different research areas.
To model the complex spatial and functional cellular interactions within the MVU, primary murine MMEC can be cultivated in more complex 3D cell culture systems together with other cell types. Prospectively, it should also be possible to generate MVU organoids as it has been described for other tissues before 14. However, successful isolation of primary murine MMEC is inevitable for aiming at this next step.

Via bloodstream, cells and organs are supplied with oxygen, substrates and other necessary molecules. This interchange takes place in capillaries, the smallest vessels. Capillaries are formed by an inner endothelial cell (EC) layer whose integrity remains a prerequisite to successful regulation of muscle homeostasis between the intravascular and interstitial space. To ensure a selective transition of soluble factors and cells, EC constitute a monolayer interconnected by tight and adherens junctions 1. Besides its role as barrier for nutrients or metabolic products, EC regulate the recruitment of leukocytes in inflammatory processes. Inflammation or tissue damage leads to an up-regulation of adhesion molecules on the EC surface and production of chemokines facilitating leukocyte attachment and transmigration into the target tissue 2. Consequently, EC are critically involved in the regulation of inflammatory processes such as the defense against pathogens or tissue repair.
A dysfunction of EC is directly associated with vascular diseases, chronic kidney failure, venous thrombosis severe pathogen infections. Furthermore, EC are virtually always involved in organ-specific autoimmunity such as diabetes mellitus or multiple sclerosis 3. The barrier function between blood stream and organs is therefore controlled by a concerted interplay of different cell types. In the skeletal muscle microvascular endothelial cells (MMEC) together with muscle cells, fibroblasts and pericytes form a functional unit, the &#34;myovascular unit&#34; (MVU). Therefore, a dysfunction of the MVU might play a critical role in the pathophysiology of myopathies. However, a deeper understanding of these regulatory mechanisms is still missing and currently precludes the identification of new, urgently needed, therapeutic targets in myopathies.
To investigate the complex physiological and pathophysiological mechanisms, animal models are commonly used. However, in vitro models offer the advantage to focus on the subject of interest by excluding a variety of confounding factors. To investigate processes in vitro it is necessary to isolate pure and viable primary cells. In contrast to cell lines, primary cells isolated from transgenic animals enable to investigate the consequences of genetic modifications in vitro.
Here, a method to isolate primary murine MMEC is described by using mechanical and enzymatic dissociation followed by magnetic activated cell sorting techniques (MCS) for purification. For this purpose, magnetic beads against specific surface markers are used. Platelet endothelial cell adhesion molecule-1 (PECAM1, CD31) is mainly expressed on EC and can be used to enrich this cell type. To warrant high cell purity, cells of hematopoietic origin are excluded by a negative selection for protein tyrosine phosphatase receptor type C (PTPRC, CD45). Further, quality controls, cultivation of primary murine MMEC, potential applications and limitations as well as special considerations are presented.

<table>
	<tbody>
		<tr>
			<td>0,25% Trypsin-EDTA</td>
			<td>Thermo Fisher</td>
			<td>25200-056</td>
			<td>ready to use</td>
		</tr>
		<tr>
			<td>ACK buffer</td>
			<td></td>
			<td></td>
			<td>150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in water at a pH of 7.3</td>
		</tr>
		<tr>
			<td>Anti-mouse CD31-FITC (clone MEC13.3)</td>
			<td>Biolegend</td>
			<td>102506</td>
			<td>Isotype control: FITC Rat IgG2a, &#954; Isotype Ctrl</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45-PE (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103106</td>
			<td>Isotype control: PE Rat IgG2b, &#954; Isotype Ctrl</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Basic fibroblast growth factor</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 MicroBeads mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-418</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase-Dispase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td>Collagenase from V. alginolyticus, Dispase from B. polymyxa</td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated Multiple 6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated anti-rat IgG antibody</td>
			<td>dianova</td>
			<td>712-166-153</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (ProLong Gold antifade reagent with DAPI)</td>
			<td>Thermo Fisher</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Desoxyribonuclease</td>
			<td>Sigma Aldrich</td>
			<td>D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas</td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonat treated water</td>
			<td>Thermo Fisher</td>
			<td>AM9916</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, containing Glutamin Supplement and pyruvate</td>
			<td>Thermo Fisher</td>
			<td>31966-021</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>dNTP Mix (10 mM)</td>
			<td>Thermo Fisher</td>
			<td>R0192</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Sarstedt</td>
			<td>551,579</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 70 &#956;m Cell Strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>FC buffer</td>
			<td></td>
			<td></td>
			<td>0.1% BSA, 0.2% NaN3, 2 mM EDTA</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Fetal calf serum</td>
		</tr>
		<tr>
			<td>Fixable Viability Dye eFluor780</td>
			<td>Thermo Fisher</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (serrated, straight, 12 cm)</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (serrated, straight, 12 cm)</td>
			<td>Fine Science Tools</td>
			<td>11009-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe 100 Solo 1ml (Omnifix)</td>
			<td>Braun</td>
			<td>9161708V</td>
			<td></td>
		</tr>
		<tr>
			<td>large magnetiv columns (LS columns)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>for CD45-MACS-step</td>
		</tr>
		<tr>
			<td>MCS buffer</td>
			<td></td>
			<td></td>
			<td>0.5% BSA, 2 mM EDTA in PBS at a pH of 7.2</td>
		</tr>
		<tr>
			<td>Medium magnetic column (MS column)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>for CD31-MACS-step</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Thermo Fisher</td>
			<td>R0581</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Phosphate buffered saline, ready to use</td>
		</tr>
		<tr>
			<td>PCR buffer (5x)</td>
			<td>Thermo Fisher</td>
			<td>EP0742</td>
			<td>in a kit with the reverse transcriptase</td>
		</tr>
		<tr>
			<td>Pecam1 rat &#945;-mouse</td>
			<td>SantaCruz</td>
			<td>Sc-52713</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>primary murine muscle cells</td>
			<td>celprogen</td>
			<td>66066-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer Cdh15 (M-Cadherin)</td>
			<td>Thermo Fisher</td>
			<td>Mm00483191_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Cldn5 (claudin-5)</td>
			<td>Thermo Fisher</td>
			<td>Mm00727012_s1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Ocln (occludin)</td>
			<td>Thermo Fisher</td>
			<td>Mm00500912_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Pax-7</td>
			<td>Thermo Fisher</td>
			<td>Mm01354484_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Tjp-1 (Zonula occludens 1)</td>
			<td>Thermo Fisher</td>
			<td>Mm00493699_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer 18s rRNA (Eukaryotic endogenous control)</td>
			<td>Thermo Fisher</td>
			<td>4310893E</td>
			<td>VIC labeled</td>
		</tr>
		<tr>
			<td>qPCR buffer (Maxima Probe/ROX qPCR Master Mix (2X)</td>
			<td>Thermo Fisher</td>
			<td>K0231</td>
			<td>2 x 1,25 mL; for 200 reactions each</td>
		</tr>
		<tr>
			<td>Random mixture of single-stranded primer</td>
			<td>Thermo Fisher</td>
			<td>SO142</td>
			<td>Random Hexamer Primer</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase (200 U/&#956;L) + PCR buffer (5x)</td>
			<td>Thermo Fisher</td>
			<td>EP0742</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase Inhibitor (40 U/&#956;L)</td>
			<td>Thermo Fisher</td>
			<td>EO0381</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor (cutting edge 23 mm, sharp/sharp) )</td>
			<td>Fine Science Tools</td>
			<td>14088-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor (cutting edge 42 mm, sharp/blunt)</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Speed Coating solution</td>
			<td>PeloBiotech</td>
			<td>PB-LU-000-0002-00</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

One day after isolation, primary murine MMEC and residual other cells form conglomerates and adhere to the bottom of culture dishes (Figure 1A day 1). From day 7 on, flat and elongated cells can be observed. However, contamination of other, mostly spheroid cells, is still visible (Figure 1A day 7). Thus, another cycle of CD31 positive selection via MCS is required. Hereafter, primary murine MMEC proliferate to a density of approximately 80&#8211;90%. Upon confluence they typically form a non-overlapping monolayer of longitudinally aligned cells (Figure 1A day 14). Proliferation stops upon confluence due to contact-inhibition. After 14 days about 5&#8211;10 x 105 cells can be used for further investigations.
Quality control via flow cytometry using fixable viability dye FVD780 (to stain for dead cells), PECAM1 (CD31) and PTPRC (CD45, as marker for cells of hematopoietic origin) showed values for both viability and purity ranging around 70% each for cells immediately after the isolation (Figure 2A). Cells cultivated after another CD31 positive selection via MCS, showed satisfying values for purity as well as for viability ranging up to 95% each (Figure 2B).
To evaluate the accuracy of the selection steps, obtained cells were further investigated for gene expression of the muscle satellite cell marker genes paired box protein 7 (Pax7) and M-cadherin (Cdh15) on mRNA level by quantitative PCR (qPCR). Primary murine MMEC (pmMMEC) and differentiated primary murine muscle cells (pmMC) were used as negative and positive control, respectively. Murine muscle cells were commercially purchased. As expected, only the CD45- CD31- fraction as well as the pmMC expressed Pax7 and Cdh15, whereas CD45- CD31+ and the primary murine MMEC were negative for these markers (Figure 2C).
EC derived from capillaries in the endomysium of skeletal muscle express tight junction proteins 4,5. By using qPCR, the expression of claudin-5 (Cld5), occludin (Ocln) and zonula occludens-1 (Tjp1 or ZO1) of confluent primary murine MMEC after the second CD31 MCS step was evaluated. PmMC were used as controls (Figure 2D). Primary murine MMEC expressed high levels of Cld5, Ocln and Tjp1, whereas pmMC only show low expression of Tjp1. Furthermore, immunofluorescence staining as quality control confirmed surface expression of the endothelium-specific marker PECAM1 in primary murine MMEC (Figure 2E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58901/58901fig1.jpg" /> 
Figure 1: Morphology of primary murine MMEC. (A) Representative image of cultured primary murine MMEC by phase contrast microscopy from days 1 to 14. d1 = Day 1, d7= Day 7, d14 = Day 14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58901/58901fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58901/58901fig2v2.jpg" /> 
Figure 2: Quality control of primary murine MMEC. Gating strategy for flow cytometric analysis of primary murine MMEC immediately after isolation (A) and on day 15 (B) using (I) FSC/SSC, (II) FVD780 and (IIIa) CD45, (IIIb) CD31 (dashed lines = isotype control). (C) Expression level of Pax7 and M-Cadherin (Cdh15) in CD45- CD31-, CD45- CD31+ fractions after MCS isolation as well as in primary murine MMEC (pmMMEC) and primary murine muscle cells (pmMC). Expression levels are shown as &#916;Ct values (sample - 18S rRNA), n.d. = not determined. (D) Expression level of tight junction proteins claudin-5 (Cldn5), occludin (Ocln) or zonula occludens-1 (Tjp1 or ZO-1) of pmMMEC and pmMC, shown as &#916;Ct values. (E) Immunofluorescence staining for PECAM1 (red) in cultivated primary murine MMEC 24 h after second CD31 positive selection. Nuclear staining with DAPI-containing (blue) mounting medium; left = anti-PECAM1/DAPI, right: negative control/DAPI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58901/58901fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells at JoVE.com

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

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 ...

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Nanyang Technological University

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JoVE Journal

Neuroscience

10.1K Views.  University of Muenster. 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 "myovascular unit".

Video: Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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 ...

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 ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...