Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Stanford University School of Medicine.
NOTE: Animals are under anesthetization with 3% vaporizer isoflurane and 3% supply of O2 gas. Use of vet ointment on eyes may help to prevent dryness while under anesthesia.
1. Cell Preparation
<ol>
	<li>Grow human endothelial and pericyte cell cultures in 100 mm plates with 10 ml of the appropriate media. Replace 10 ml of fresh medium every 2 - 3 days until cells reach 80% confluency.
	<ol>
		<li>Culture endothelial cells (ECs) in complete endothelial cell media (ECM) supplemented with company supplied 5% fetal bovine serum (FBS), 10% penicillin/streptomycin and 10% endothelial cell growth supplement.</li>
		<li>Culture pericytes in complete pericyte media (PM) supplemented with company supplied 2% FBS, 10% penicillin/streptomycin and 10% pericyte growth supplement.</li>
	</ol>
	</li>
	<li>Prepare cell mixtures
	<ol>
		<li>Calculate the required number of cell for the experimental and control groups. 
		NOTE: Each plug in the experimental group contains one million (1 x 106) human ECs and two hundred thousand (2 x 105) pericytes. For the control group, each plug contains 1.2 million human ECs only. The negative control group has matrix gel only. Each group should include at least four plugs, which would require two mice as plugs can be injected into each side of a mouse&#39;s lower back. Three plugs would serve as triplicate for the experiment and the fourth could be used in case one fails. Prepare 25% extra cells to match the extra volume of gel.</li>
		<li>Collect and count cells from culture plates.
		<ol>
			<li>To detach cells, remove media and wash once with room temperature 1x phosphate buffered saline (PBS). Remove PBS, add 1 ml 0.25% Trypsin/2.21 mM EDTA, and incubate for 1 min. Wash cells off the plate by adding 2 ml medium and collect in a 15 ml tube.</li>
			<li>Count cell using a hemocytometer according to the manufacturer&#39;s instructions.</li>
			<li>Centrifuge the appropriate number of cells into a pellet at 400 x g for 5 min. For the experimental group, centrifuge both ECs and pericytes down together into one pellet.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Matrix Gel Preparation
<ol>
	<li>Thaw out matrix gel completely at 4 &#176;C for 4 hr or overnight to obtain a homogenous solution. No mixing is required.</li>
	<li>Aliquot matrix gel in 1.5 ml tubes and store at 4 &#176;C.</li>
	<li>Pre-chill sterile 1.5 ml tubes and 28 G 1 cc insulin syringes at 4 &#176;C. Caution: Keep matrix gel on ice at all times.</li>
	<li>Mix cold matrix gel with bFGF at a volume ratio of 1:100 (final concentration of bFGF= 0.5 &#956;g/ml; bFGF stock solution= 50 &#956;g/ml) After mixing matrix gel solution by pipetting up and down several times, incubate on ice for 30 - 60 min before mixing with the cells in step 3.1. 
	NOTE: Thaw the stock bFGF before mixing. Calculate the number of plugs needed for injection. For example, each plug requires 200 &#956;l matrix gel. Therefore, prepare 25% extra gel.</li>
</ol>
3. Mix Matrix Gel with Cells
<ol>
	<li>For each group, resuspend the cell pellet (containing appropriate cell number) with the calculated volume of matrix gel containing bFGF. Mix gently to avoid foaming and leave in the 15 ml tube on ice until mice are prepared for injection.</li>
</ol>
4. Mouse Preparation
<ol>
	<li>Anesthetize 1&#160;SCID mice for 5 min in 3% Isoflurane plus 3% O2 in an induction chamber. Focus on one experimental group, or 2 mice, at a time.</li>
	<li>Remove the&#160;mouse from the induction chamber, place on a heating pad ventral side down and quickly attach a snout nozzle to the mouse&#39;s face via a non-rebreathing system to supply anesthesia throughout the injection procedure. Switch the gas flow from the induction chamber to the non-rebreathing system and lower the gas pressure to 1.5% Isoflurane and 1.5% O2.</li>
	<li>Remove hair from both lateral hind regions (around 1 cm diameter) with a shaver or hair removal cream.</li>
	<li>Wipe the skin area with a 70% alcohol pad.</li>
	<li>Return the mouse to the induction chamber and repeat steps 4.2 - 4.4 with the second mouse.</li>
</ol>
5. Matrix Gel Injection
<ol>
	<li>Prepare one mouse for injection by attaching to the non-rebreathing system and placing on the heating pad ventral side down.</li>
	<li>Load the matrix gel into a pre-chilled 28 G 1 cc insulin syringe one mouse&#160;at a time.
	<ol>
		<li>Load the complete 500 &#956;l&#160;volume of matrix gel for two&#160;plugs (200 &#956;l per plug plus 25% extra) into the syringe.</li>
		<li>Make sure to inject the matrix gel into the mice within 3 min of loading the cells into the syringe to prevent the cells from settling to the side of the syringe and to prevent matrix gel from solidifying inside of the syringe.</li>
	</ol>
	</li>
	<li>Lift the back skin to locate subcutaneous space, then inject 200 &#956;l of the matrix gel slowly and evenly into the subcutaneous space of the back posterior of the rib cage.</li>
	<li>Remove the injection needle slowly, being careful to prevent matrix gel from leaking out of the injection site. A bump will form at the site of injection. Wipe injection site with an alcohol pad.</li>
	<li>Repeat injection steps 5.2 - 5.5 on the other side of mouse&#39;s back, then leave the mouse on its back for 1 min to allow gelation of the matrix gel on the heat pad.</li>
	<li>Outline both bumps using a permanent marker to identify the bump after 14 days. After some hair grows back, it will be easier to find the bump.</li>
</ol>
6. Matrix Gel Plug Isolation: 14 Days after Injection
<ol>
	<li>Euthanize the mice humanely by exposing the animals to &#62;5 min of carbon dioxide inhalation followed by cervical dislocation to assure death.</li>
	<li>Remove the matrix gel plug from the mouse&#39;s back.
	<ol>
		<li>Gently remove regrown hair around injection site where the plug is located by repeating step 4.3. The marker line indicates the size of original bump under the skin.</li>
		<li>Excise the plug along with the surrounding skin and muscle layers attached on the top and bottom sides of the plug, respectively.
		<ol>
			<li>Using fine surgical scissors, make an incision on the marked border of the plug that is perpendicular to the skin and cut through to the muscle underneath the plug. Then, carefully cut around the periphery of the plug along the marked border.</li>
			<li>Remove the plug, keeping it sandwiched between the skin and muscle layers. Immediately rinse in a small beaker with 1x PBS to wash away excess blood. The plug is now ready to be fixed (next step, 6.3).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fix the plug in 4% paraformaldehyde.
	<ol>
		<li>Place the entire plug, including the skin and muscle layers, in a 50 ml tube with 25 ml of 4% paraformaldehyde for 24 hr at room temperature without rocking. The next day, transfer the plug into 25 ml of 70% ethanol for another 24 hr before paraffin embedding. 
		NOTE: Handle paraformaldehyde with caution. Wear gloves.</li>
	</ol>
	</li>
</ol>
7. Paraffin Process the Matrix Gel Plug
<ol>
	<li>Prepare the plug for paraffin embedding.
	<ol>
		<li>Orient the plug as shown in Figure 1a; place the plug in a tissue cassette so that the side of the plug is facing up and both the skin and muscle layers are visible across the surface of the tissue block that will be cut first during sectioning.</li>
	</ol>
	</li>
	<li>Paraffin embed the plug according to standard paraffin embedding protocols13.</li>
	<li>Cut 8 - 10 &#956;m thick sections off of the tissue block and mount onto microscope slides according to standard paraffin sectioning protocols13.</li>
	<li>Store the slides in a cool dry place at room temperature until ready for staining.</li>
</ol>
8. Stain Sections with Hematoxylin and Eosin Stain (H&#38;E)13
<ol>
	<li>De-paraffinize the tissue sections by passing the slides through 100% xylene twice, 100% ethanol, 95% ethanol, 70% ethanol and lastly 1x PBS, each for 5 min at room temperature. Leave slides in 1x PBS until the next step.</li>
	<li>Pipette 100 &#956;l H&#38;E solution onto the section and incubate for 1 - 5 min, or until there is clear contrast between blue nuclei and pink cytoplasm in a cell as viewed under an upright light microscope at 10X magnification. Be careful not to let any H&#38;E solution touch the objective.</li>
	<li>Wash H&#38;E solution off of the slide by dunking the slide in a large beaker of tap water, then place the slide in a rack in the beaker and flush with running water for 2 min.</li>
	<li>Move to step 9.8 to mount the slides.</li>
</ol>
9. Stain Other Slides for Human Capillaries and Pericytes
<ol>
	<li>De-paraffinize the tissue sections by passing the slides through 100% xylene twice, 100% ethanol, 95% ethanol, 70% ethanol and lastly 1x PBS, each for 5 min at room temperature. Leave slides in 1x PBS until the next step.</li>
	<li>Boil the sections in antigen retrieval solution.
	<ol>
		<li>In a 2 L beaker, prepare boiling antigen retrieval solution using 1x citrate buffer (pH 7.0) at 95 &#176;C on a heating block.</li>
		<li>Once boiling, place the slides in a metal rack and submerge in the boiling antigen retrieval solution for 20 min.</li>
		<li>Carefully remove the beaker from the heating block with the slides still submerged and allow the solution to slowly return to room temperature for about 30 min.</li>
		<li>Place the slides in 1x PBS until next step.</li>
	</ol>
	</li>
	<li>Block the sections in blocking buffer (1x PBS, 5% Goat Serum, 0.3% Triton X-100).
	<ol>
		<li>Draw a hydrophobic circle around the edges of each section with a PAP pen.</li>
		<li>Place the slides in a humidity tray with distilled water at the bottom of the tray.</li>
		<li>Pipette 100 &#956;l blocking buffer onto the sections making sure all of the tissue is covered by fluid (may require more than 100 &#956;l).</li>
		<li>Incubate sections with blocking buffer for 1 hr.</li>
	</ol>
	</li>
	<li>Incubate sections with primary antibody.
	<ol>
		<li>Prepare one solution with two primary antibodies to probe each section for CD31 (ECs) and smooth muscle actin (pericytes) diluted in dilution buffer (1x PBS, 1%BSA, 0.3% Triton X-100). 
		NOTE: Here, anti-CD31 concentration is 1:50 and anti-smooth muscle actin concentration is 1:300.</li>
		<li>Incubate the sections overnight at 4 &#176;C with 100 &#956;l primary antibodies in a humidity tray containing distilled water.</li>
	</ol>
	</li>
	<li>Wash the slides three times in 1x PBST (1x PBS + 0.1% Tween20) for 5 min each wash.</li>
	<li>Incubate sections with secondary antibody.
	<ol>
		<li>Prepare a solution with a fluorophore conjugated goat anti-rabbit secondary antibody to recognize the anti-CD31 rabbit antibody diluted in dilution buffer.
		<ol>
			<li>NOTE: The smooth muscle actin primary antibody is already conjugated with a CY3 fluorophore, so no secondary antibody staining is needed. Goat-anti rabbit secondary antibody concentration is 1:250.</li>
		</ol>
		</li>
		<li>Incubate the sections for 1 hr with 100 &#956;l secondary antibody in the humidity tray containing distilled water.</li>
	</ol>
	</li>
	<li>Wash the slides three times in 1x PBST for 5 min each wash.</li>
	<li>Mount the slides.
	<ol>
		<li>Pipette 2 - 3 drops (~ 40 - 60 &#956;l) of antifade mounting solution with DAPI nuclear staining onto each section.</li>
		<li>Gently place a no. 1.5 cover slip over the sections and mounting solution, allowing the fluid to spread out over the sections and to the edges of the cover slip. Gently use a finger to press out air bubbles that may be located over the section.</li>
		<li>Let the slides dry for at least 1 hr before handling.</li>
	</ol>
	</li>
</ol>
10. Quantify Capillary Density and Structure14
<ol>
	<li>Count the number of capillaries in H&#38;E stained sections, expressed as #/mm2.
	<ol>
		<li>Acquire photos of four different fields on an upright light microscope under 40X magnification. 
		NOTE: Capillaries are identified as tubes filled with red blood cells.</li>
		<li>Alternatively, instead of manually counting capillaries, use image analysis software (e.g., Wimasis Image Analysis) to quantify various angiogenesis parameters such as vessel number, average vessel length and diameter, and branch points.</li>
	</ol>
	</li>
	<li>Analyze capillary structure in immunofluorescent stained sections.
	<ol>
		<li>Acquire photos of four different fields on an inverted fluorescent microscope with FITC and TxRed filter cube.
		<ol>
			<li>Use a FITC cube to image ECs by excitation at 488 nm wavelength and use a TxRed cube to image pericytes by excitation at 594 nm wavelength.
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			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Risau, W. <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+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Ribatti, D., Nico, B., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pericytes+in+angiogenesis.">The role of pericytes in angiogenesis.</a> Int J Dev Biol. 55 (3), 261-268 (2011).</li><li>Koh, W., Stratman, A. N., Sacharidou, A., Davis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+three+dimensional+collagen+matrix+models+of+endothelial+lumen+formation+during+vasculogenesis+and+angiogenesis.">In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.</a> Methods Enzymol. 443, 83-101 (2008).</li><li>Liu, Y., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-specific+activation+of+Src+and+Rho+initiates+capillary+morphogenesis+of+endothelial+cells.">Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells.</a> FASEB J. 18 (3), 457-468 (2004).</li><li>Grant, D. S., 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=Interaction+of+endothelial+cells+with+a+laminin+A+chain+peptide+(SIKVAV)+in+vitro+and+induction+of+angiogenic+behavior+in+vivo.">Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.</a> J Cell Physiol. 153 (3), 614-625 (1992).</li><li>Madri, J. A., Pratt, B. M., Tucker, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+modulation+of+endothelial+cells+by+transforming+growth+factor-beta+depends+upon+the+composition+and+organization+of+the+extracellular+matrix.">Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix.</a> J Cell Biol. 106 (4), 1375-1384 (1988).</li><li>Brooks, P. C., Montgomery, A. M., Cheresh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+10-day-old+chick+embryo+model+for+studying+angiogenesis.">Use of the 10-day-old chick embryo model for studying angiogenesis.</a> Methods Mol Biol. 129, 257-269 (1999).</li><li>Yuan, K., 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=Activation+of+the+Wnt/planar+cell+polarity+pathway+is+required+for+pericyte+recruitment+during+pulmonary+angiogenesis.">Activation of the Wnt/planar cell polarity pathway is required for pericyte recruitment during pulmonary angiogenesis.</a> Am J Pathol. 185 (1), 69-84 (2015).</li><li>Johns, A., Freay, A. D., Fraser, W., Korach, K. S., Rubanyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+estrogen+receptor+gene+prevents+17+beta+estradiol-induced+angiogenesis+in+transgenic+mice.">Disruption of estrogen receptor gene prevents 17 beta estradiol-induced angiogenesis in transgenic mice.</a> Endocrinology. 137 (10), 4511-4513 (1996).</li><li>Chen, C. W., 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=Human+pericytes+for+ischemic+heart+repair.">Human pericytes for ischemic heart repair.</a> Stem Cells. 31 (2), 305-316 (2013).</li><li>Richards, O. C., Raines, S. M., Attie, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+blood+vessels,+endothelial+cells,+and+vascular+pericytes+in+insulin+secretion+and+peripheral+insulin+action.">The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action.</a> Endocr Rev. 31 (3), 343-363 (2010).</li><li>Ricard, N., 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=Increased+pericyte+coverage+mediated+by+endothelial-derived+fibroblast+growth+factor-2+and+interleukin-6+is+a+source+of+smooth+muscle-like+cells+in+pulmonary+hypertension.">Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension.</a> Circulation. 129 (15), 1586-1597 (2014).</li><li>Buchwalow, I. B., Bocker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistochemistry: Basics and Methods. , 1-153 (2010).</li><li>Tata, D. A., Anderson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+investigation+of+capillary+structure.">A new method for the investigation of capillary structure.</a> J Neurosci Methods. 113 (2), 199-206 (2002).</li></ol>

No competing financial interests enclosed.

The matrix gel plug assay has proven to be a convenient and powerful method to evaluate gene regulation in angiogenesis, angiogenic and antiangiogenic compounds in vivo, and to supplement in vitro tests. Here, we describe in detail a novel matrix gel plug assay of human angiogenesis that investigates the interaction between endothelial cells and pericytes during vessel formation.
There are a few novel and critical steps in this protocol. Cells in low passage (passage 1 - 4) a...

Angiogenesis is the process by which new blood vessels are formed from a pre-existing vascular network1 and is the focus of ongoing research across many areas ranging from normal development to disease. This dynamic process involves the proliferation and migration of endothelial cells (ECs) and recruitment of pericytes to construct a vascular tube that is directed toward the site that needs oxygen and nutrient delivery2. To study this process requires an equally dynamic assay, most importantly one that can recapitulate the three-dimensional nature of tube formation. In vitro 3D matrix assays have been developed to address this need and have worked well to allow researchers to define the discrete steps in space and time in which angiogenesis takes place3,4,5,6. However, these in vitro 3D matrix models are limited to studying non-perfused vessels and therefore lack critical components pertinent to the angiogenesis process (e.g., circulating growth and inhibitory factors, unnatural tension/forces across the vascular bed) and fail to simulate the complex environment present in live tissue. To address this limitation, several in vivo angiogenesis assays have been developed7, including the matrix gel plug assay which will be the focus of our report8,9.
The matrix gel plug assay is a well-established in vivo angiogenesis assay that appeals to researchers as it provides a robust platform to test the roles of different cells and substances in angiogenesis. Matrix gel is a commercially available basement membrane solution that is secreted by the Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell line that solidifies into a gel-like material at 37 &#176;C. The matrix gel can be mixed with cells and/or substances, such as growth factors, and injected subcutaneously into the mouse. The host ECs will invade the plug over 14 days, form a vascular network, and become perfused with the host&#39;s blood. To date, matrix gel plug assays have focused exclusively on the study of endothelial cell behavior during angiogenesis, however, to the best of our knowledge no effort has yet been made to determine whether this assay can be used to co-culture endothelial cells and pericytes to study how these two cell types interact during angiogenesis. Specifically, understanding the relationship between ECs and pericytes is valuable for studying diseases where blood vessel loss is pathologic, including microvascular ischemia and peripheral vascular disease10,11,12.
Here, we describe a protocol that introduces human-derived pericytes to the matrix gel mixture along with human ECs and fibroblast growth factor (bFGF). This mixture can then be injected subcutaneously in the dorsum of SCID mice to allow formation of fully functional, pericyte coated, hybrid vessels. Our protocol describes how to prepare matrix gel plugs containing human ECs either with or without human pericytes, placement into SCID mice and how to analyze the histological sections for critical angiogenesis endpoints.

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">PBS (Phosphate buffered saline)</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.25% Trypsin/0.53mM EDTA</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Endothelial Cell Media (ECM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1001</td>
			<td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Pericyte Media (PM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1201</td>
			<td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary microvascular endothelial cells</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is also available commercially. Cells used at passage 1-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary pericytes</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is not available commercially, but brain paricytes are.&#160; Cells used at passage 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">bFGF (basic Fibroblast Growth Factor)</td>
			<td style="width:115px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">356237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">28G 1cc Insluin Syringe</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">329410</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">SCID (Severe Combined Immune Deficiency) mice</td>
			<td style="width:115px;">The Jackson Laboratory</td>
			<td>5557</td>
			<td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">1.5mL microcentrifuge tubes, sterile</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">15 mL screw top tubes, sterile</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">PAP pen</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">8899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">hemocytometer</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Nair hair removal cream</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">anti-human CD31 primary antibody</td>
			<td style="width:115px;">LifeSpan Biosciences</td>
			<td>LS-B4737</td>
			<td>working solution is 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td>
			<td style="width:115px;">Sigma</td>
			<td>C6198</td>
			<td>working solution is 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit secondary antibody; 488/green</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A-11008</td>
			<td>working solution is 1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Prolong Gold DAPI solution</td>
			<td style="width:115px;">Cell Signaling</td>
			<td style="width:119px;">8961S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">microscope slides</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">48300-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">no. 1.5 cover slips</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-544-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">citrate buffer 10x</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">21545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">extra fine surgical scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Formalin (paraformaldehyde)</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">245-685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tissue cassettes</td>
			<td style="width:115px;">Simport</td>
			<td>M492-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">goat serum</td>
			<td style="width:115px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

Representative H&#38;E and immunofluorescent staining of matrix gel plug sections are shown in Figure 2. Sections from EC only plugs display some vessels that are mostly not perfused with blood (Figure 2 top left, black arrows) whereas plugs containing both ECs and pericytes display several perfused vessels with larger diameters and complete pericyte coverage, as evidenced by positive SMA staining immediately adjacent to CD31-positive ECs. These results s...

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

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9.6K Views.  Stanford University. We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Video: In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...