This work was supported by funds from the Italian Ministry of the University and Research (ex 60% 2021 and 2022) to R. P. and by grants from the Italian Cystic Fibrosis Foundation (&#65279;FFC#23/2014) and from the Italian Ministry of Health (&#65279;L548/93) to M. R.

This study was approved, and the protocol followed the guidelines of the human research ethics committee of the University of Chieti-Pescara (#237_2018bis). Figure 1 illustrates the isolation of endothelial cells from segments (1-3 cm long) of pulmonary parenchyma or pulmonary artery from deidentified human subjects (with written consent) undergoing thoracic surgery for various reasons, such as pneumothorax or lobectomy. In this latter case, the surgeons also collected a pulmonary artery segment. Notably, the surgeons were accurately instructed to collect cancer-free samples. The presented protocol was optimized to obtain the greatest possible yield and purity.
1. Experimental preparation
<ol>
	<li>Collagenase reconstitution
	<ol>
		<li>Dissolve collagenase powder in phosphate-buffered saline without CaCl2 and MgCl2 (PBS&#8722;&#8722;, see Table of Materials) at a 2 mg/mL concentration, and filter the solution using a 0.22 &#181;m pore filter.</li>
		<li>Prepare 5 mL aliquots, and store them at &#8722;20 &#176;C. Thaw and preheat the aliquots to 37 &#176;C prior to use.</li>
	</ol>
	</li>
	<li>Plate coating
	<ol>
		<li>For plate coating, pipette 1.5% gelatin solution or 50 &#181;g/mL fibronectin (see Table of Materials) into the culture plate (500 &#181;L is enough to cover the surface of each well of a 6-well plate), and incubate for 1 h at 37 &#176;C.</li>
		<li>After incubation, remove the excess gelatin, and wash the wells with PBS&#8722;&#8722;. Aspirate the PBS&#8722;&#8722;, and let the plate dry in a sterile hood. 
		NOTE: For gelatin reconstitution, dissolve the powder in water to make a 1.5% solution, then autoclave it, and store at 4 &#176;C. Gelatin at 1.5% is not liquid at 4 &#176;C; therefore, it must be warmed up prior to plate coating. Alternatively, any commercial gelatin solution suitable for cell cultures can be used for the plate coating.</li>
	</ol>
	</li>
</ol>
2. Sample preparation
<ol>
	<li>Lung parenchyma
	<ol>
		<li>Wash the collected samples by immersing them in a 50 mL tube containing PBS&#8722;&#8722;.</li>
		<li>Transfer the samples into a sterile Petri dish, and manually chop the sample using surgical scissors (optimal size: &#62;2 cm) into small fragments of about 3-4 mm each.</li>
	</ol>
	</li>
	<li>Artery segment(s)
	<ol>
		<li>Wash the collected samples by immersing them in a 50 mL tube containing PBS&#8722;&#8722;.</li>
		<li>Transfer it into a sterile Petri dish without chopping, as fragmentation will increase the surface area of the cross-section of the artery segment, thus increasing the possibility of isolating non-ECs.</li>
	</ol>
	</li>
</ol>
3. Enzymatic digestion
<ol>
	<li>Wash the diced lung parenchyma or the pulmonary artery segment/s twice with PBS&#8722;&#8722;. This step will remove a large part of the residual blood.</li>
	<li>Place the samples in 15 mL tubes, and incubate with 5 mL of type 2 collagenase (see Table of Materials) for 10 min at 37 &#176;C and 5% CO2. During this incubation, the degradation of the extracellular matrix releases single cells and cell aggregates.</li>
</ol>
4. Recovery of the digested cells
<ol>
	<li>Place a sterile steel/metal strainer (i.e., a tea strainer with a ~1 mm mesh size) on the top of a 50 mL collection tube.</li>
	<li>Pour the entire contents from the 15 mL tube onto the strainer, gently massage the digested tissue with a spatula, and rinse the sample with PBS&#8722;&#8722;&#160;until the collection tube is filled. 
	&#8203;NOTE: This step will eliminate large tissue fragments on the millimeter scale, ensuring greater efficiency of the subsequent filtration steps.</li>
</ol>
5. Non-EC removal by filtration
<ol>
	<li>Filter the outflow collected in step 4.2 using a cell strainer with a 70 &#181;m mesh size, and collect the outflow in a fresh 50 mL tube.</li>
	<li>Then, use a cell strainer with a 40 &#181;m mesh size, and collect the outflow in a fresh 50 mL tube. Label this tube as &#34;tube 1&#34;. 
	&#8203;NOTE: These filtrations in step 5.1 and step 5.2 will remove large cell aggregates, which are often composed of mixed cell populations.</li>
</ol>
6. Sedimentation of cell clusters and cell seeding
<ol>
	<li>Centrifuge the samples at 30 x g for 5 min at room temperature to sediment the cell clusters.</li>
	<li>Carefully remove the supernatant using a sterile pipette (do not pour), and place it in a fresh 50 mL tube (&#34;tube 2&#34;).</li>
	<li>Suspend the pellet in &#34;tube 1&#34;, and fill the tube up to 50 mL with PBS&#8722;&#8722;. Fill &#34;tube 2&#34; up to 50 mL with PBS&#8722;&#8722;. 
	NOTE: From this step onward, both tubes are treated in the same way.</li>
	<li>Centrifuge the samples at 300 x g for 5 min at room temperature.</li>
	<li>Suspend the pellets with 2 mL of growth medium (see Table of Materials), and seed the suspension in two separate wells of a pre-coated 6-well plate (step 1.2). 
	&#8203;NOTE: From this step, feed the cells every 2 days with the growth medium until they reach confluency (approximately 1 week, depending on the sample size) in order to obtain a reasonable number of cells for sorting.</li>
</ol>
7. Cell expansion
<ol>
	<li>Wash the cells with 2 mL of PBS with CaCl2 and MgCl2 (PBS++, see Table of Materials) to remove the residual blood cells (using PBS++ is important to avoid cell detachment).</li>
	<li>Remove the PBS++, and add fresh culture medium.</li>
	<li>Repeat step 7.1 and step 7.2 until the cells reach confluency (approximately 1 week, depending on the sample size).</li>
</ol>
8. Cell sorting
<ol>
	<li>Detach the cells using 500 &#181;L of trypsin-EDTA (0.05%, see Table of Materials), centrifuge, and resuspend the pellet with 190 &#181;L of PBS&#8722;&#8722;.</li>
	<li>Add 10 &#181;L of a fluorescent conjugate anti-human CD31-FITC antibody (1:20 dilution, see Table of Materials), and incubate the cell suspension at 4 &#176;C for 30 min.</li>
	<li>Wash the cells using 10 mL of PBS&#8722;&#8722;, centrifuge at 300 x g for 5 min at room temperature, and resuspend the pellet in 300 &#181;L of PBS&#8722;&#8722;.</li>
	<li>Isolate and collect CD31 positive (CD31+) cells by fluorescence-activated cell sorting (FACS), using a 100 &#956;m nozzle, and collect them in a tube.
	<ol>
		<li>As per the gating strategy, first define the cell morphology using the side scatter area parameters, SSC-A and FSC-A. Then, select single cells using an FSC-Area/FSC-Height or SSC-Area/SSC-Height dot plot, define the positive cells in the marked sample versus the unstained control sample, and direct the fluorescent cells into the collection tube21.</li>
	</ol>
	</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min at room temperature, and suspend the pellet in 2 mL of growth medium for seeding in the pre-coated wells of a 6-well plate.</li>
</ol>
9. Post-sorting expansion
<ol>
	<li>Two days after cell sorting, replace the conditioned medium with fresh growth medium.</li>
	<li>Repeat step 7.1 and step 7.2 every 2 days for cell expansion.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+regulation+of+vascular+tone+and+growth.">Endothelial regulation of vascular tone and growth.</a> American Journal of Hypertension. 6, 283-293 (1993).</li><li>Marki, A., Esko, J. D., Pries, A. R., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+endothelial+surface+layer+in+neutrophil+recruitment.">Role of the endothelial surface layer in neutrophil recruitment.</a> Journal of Leukocyte Biology. 98 (4), 503-515 (2015).</li><li>Crampton, S. P., Davis, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+umbilical+vein+endothelial+cells+(HUVEC).">Isolation of human umbilical vein endothelial cells (HUVEC).</a> Journal of Visualized Experiments. (3), e183 (2007).</li><li>Ganguly, A., Zhang, H., Sharma, R., Parsons, S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+umbilical+vein+endothelial+cells+and+their+use+in+the+study+of+neutrophil+transmigration+under+flow+conditions.">Isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions.</a> Journal of Visualized Experiments. (66), e4032 (2012).</li><li>Dejana, E., Corada, M., Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-to-cell+junctions.">Endothelial cell-to-cell junctions.</a> FASEB Journal. 9 (10), 910-918 (1995).</li><li>Plebani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+long-term+culture+of+human+cystic+fibrosis+endothelial+cells.">Establishment and long-term culture of human cystic fibrosis endothelial cells.</a> Laboratory Investigation. 97 (11), 1375-1384 (2017).</li><li>Totani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+endothelial+cell+dysfunction+in+cystic+fibrosis.">Mechanisms of endothelial cell dysfunction in cystic fibrosis.</a> Biochimica Et Biophysica Acta. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+pulmonary+artery+endothelial+cells+from+pulmonary+artery+catheter+balloon+tips:+considerations+for+use+in+pulmonary+vascular+disease.">Culture of pulmonary artery endothelial cells from pulmonary artery catheter balloon tips: considerations for use in pulmonary vascular disease.</a> The European Respiratory Journal. 55 (3), 1901313 (2020).</li><li>Wang, J., Niu, N., Xu, S., Jin, Z. 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The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

The multiple roles played by vascular endothelial cells in human pathophysiology make these cells an indispensable tool for in vitro pathogenetic and pharmacological studies. Since ECs from different vascular sites/organs display peculiar features and functions, the availability of healthy and diseased ECs from the organ of interest would be ideal for research purposes. For instance, HLMVECs are essential for studies on lung inflammation; therefore, a methodology for the high-yield, high-purity isolation of thes...

The vascular endothelium lines the inner surface of the blood vessels. It plays key roles in regulating blood coagulation, vascular tone, and immune-inflammatory responses1,2,3,4. Although the culture of endothelial cells (ECs) isolated from human specimens is essential for research purposes, it must be remarked that the ECs from different blood vessels (arteries, veins, capillaries) have specific functions. These cannot be fully recapitulated by human umbilical vein endothelial cells (HUVEC), which are easily available and widely used in studies on vascular endothelium pathophysiology5,6. For instance, human lung microvascular endothelial cells (HLMVECs) play key roles in lung inflammation by controlling leukocyte recruitment and accumulation4,7. Thus, an experimental setting aimed at reproducing lung inflammation with high fidelity should include HLMVECs. On the other hand, EC dysfunction can be observed in several pathologies; therefore, ECs from the patient are fundamental to building a reliable in vitro model of the disease. For instance, the isolation of fragments of ECs from the pulmonary artery (HPAECs), dissected from the explanted lungs of people affected by cystic fibrosis (CF), have enabled us to uncover mechanisms of endothelial dysfunction in this disease8,9.
Thus, protocols aimed at optimizing the isolation of ECs from different sources/organs also in disease states are essential to provide investigators with valuable research tools, particularly when these tools are not commercially available. HLMVEC and HPAEC isolation protocols have been previously reported10,11,12,13,14,15,16,17,18,19. In all cases, the enzymatic digestion of the lung specimens resulted in mixed cell populations, which were purified using ad hoc selective media and magnetic beads- or cytometric-based cell sorting. Further optimizations of these protocols must address two main issues in EC isolation: (1) cell and tissue contamination, which should be resolved at the earliest possible culture passages to minimize EC replicative senescence20; and (2) the low yield of primary EC isolates.
This study describes a new protocol for the high-yield, high-purity isolation of HLMVECs and HPAECs. This procedure can be easily applicable and give virtually pure macrovascular and microvascular ECs in a few steps.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin-EDTA 1X</td>
			<td>GIBCO</td>
			<td>25300-054</td>
			<td>Used to detach cells from the culture plates</td>
		</tr>
		<tr>
			<td>Anti CD31 Antibody, clone WM59</td>
			<td>Dako</td>
			<td>M0823</td>
			<td>Used for CD-31 staining in immunocytochemistry. Dilution used: 1:50</td>
		</tr>
		<tr>
			<td>Anti vWF Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-14029</td>
			<td>Used for von Willebrand factor staining in immunocytochemistry. Working dilution: 1:100</td>
		</tr>
		<tr>
			<td>Autoclavable surgical scissors</td>
			<td>Any</td>
			<td></td>
			<td>Used for chopping specimens</td>
		</tr>
		<tr>
			<td>Cell strainers 40 &#181;m</td>
			<td>Corning</td>
			<td>431750</td>
			<td>Used during the second filtration</td>
		</tr>
		<tr>
			<td>Cell strainers 70 &#181;m</td>
			<td>Corning</td>
			<td>431751</td>
			<td>Using during the first filtration</td>
		</tr>
		<tr>
			<td>Collagenase, Type 2</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td>Type 2 Collagenase used for enzymatic digestion. Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Conjugated anti CD31 Antibody</td>
			<td>BD Biosciences</td>
			<td>555445</td>
			<td>Used for cell sorting (1:20 dilution)</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS) with&#160; CaCl2 and MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td>Used for cell washing before medium change</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS) without CaCl2 and MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Used for washing surgical specimens and cells before trypsinization</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium MV</td>
			<td>PromoCell</td>
			<td>C-22020</td>
			<td>HLMVEC growth medium</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td>Fibronectin from human plasma used for plate coating. Working concentration: 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin, type A</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td>Used for plate coating</td>
		</tr>
		<tr>
			<td>Type A gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>g-2500</td>
			<td>Gelatin from porcine skin used for plate coating. Working concentration: 1.5%</td>
		</tr>
	</tbody>
</table>

microvascular and macrovascular endothelial cell isolation and purification from lung-derived samples

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

HLMEC isolation 
The main problem during the isolation of HLMVECs is the presence of contaminating cells since the microscopic capillaries cannot be easily separated from the stromal tissue. Therefore, achieving the highest possible purity at the earliest stages of the isolation process is crucial in order to reduce the culture passages and, thus, the cell aging. Likewise, an optimal isolation protocol should give the highest possible yield of pure HLMVECs. To achieve these goals, a new procedure wa...

Watch this Scientific Journal Video about Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples at JoVE.com

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although ...

microvascular-macrovascular-endothelial-cell-isolation-purification

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1.9K Views.  “G. d’Annunzio” University of Chieti-Pescara. Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

Video: Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...