Development of the protocols described in this paper was supported by grants from the following funding agencies: BBSRC (BB/I026359/1), Fight for Sight (1429 and 1822), The Juvenile Diabetes Research Foundation (2-2003-525), Wellcome Trust (074648/Z/04), British Heart Foundation (PG/11/94/29169), HSC R&#38;D Division (STL/4748/13) and MRC (MC_PC_15026).

<ol>
	<li>Kohner, E. M., Patel, V., Rassam, S. M. <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+blood+flow+and+impaired+autoregulation+in+the+pathogenesis+of+diabetic+retinopathy.">Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy.</a> Diabetes. 44, 603-607 (1995).</li><li>Hafez, A. S., Bizzarro, R. L., Lesk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+optic+nerve+head+and+peripapillary+retinal+blood+flow+in+glaucoma+patients,+ocular+hypertensives,+and+normal+subjects.">Evaluation of optic nerve head and peripapillary retinal blood flow in glaucoma patients, ocular hypertensives, and normal subjects.</a> American Journal of Ophthalmology. 136, 1022-1031 (2003).</li><li>Curtis, T. M., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+lesions+of+diabetic+retinopathy:+clues+towards+understanding+pathogenesis?.">Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?.</a> Eye (London). 23, 1496-1508 (2009).</li><li>Pournaras, C. J., Rungger-Br&#228;ndle, E., Riva, C. E., Hardarson, S. H., Stefansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+retinal+blood+flow+in+health+and+disease).">Regulation of retinal blood flow in health and disease).</a> Progress in Retinal and Eye Research. 27, 284-330 (2008).</li><li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+retinal+vascular+patterns.+I.+Normal+architecture.">Studies of retinal vascular patterns. I. Normal architecture.</a> Archives of Ophthalmology. 64, 904-911 (1960).</li><li>Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+physiology+of+retinal+and+choroidal+arteriolar+smooth+muscle+cells.">Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.</a> Microcirculation. 14 (1), 11-24 (2007).</li><li>Puro, D. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+heterogeneity+of+KIR+currents+in+pericyte-containing+microvessels+of+the+rat+retina:+effect+of+diabetes.">Topographical heterogeneity of KIR currents in pericyte-containing microvessels of the rat retina: effect of diabetes.</a> Journal of Physiology. 573, 483-495 (2006).</li><li>Needham, M., 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=The+role+of+K++and+Cl-+channels+in+the+regulation+of+retinal+arteriolar+tone+and+blood+flow.">The role of K+ and Cl- channels in the regulation of retinal arteriolar tone and blood flow.</a> Investigative Ophthalmology &amp; Visual Science. 55 (4), 2157-2165 (2014).</li><li>Hessellund, A., Jeppesen, P., Aalkjaer, C., Bek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+vasomotion+in+porcine+retinal+arterioles.">Characterization of vasomotion in porcine retinal arterioles.</a> Acta Ophthalmologicaogica Scandinavica. 81, 278-282 (2003).</li><li>Delaey, C., Van de Voord, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+myogenic+responses+in+isolated+bovine+retinal+arteries.">Pressure-induced myogenic responses in isolated bovine retinal arteries.</a> Investigative Ophthalmology &amp; Visual Science. 41, 1871-1875 (2000).</li><li>Kur, J., 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=Role+of+ion+channels+and+subcellular+Ca2++signaling+in+arachidonic+acid-induced+dilation+of+pressurized+retinal+arterioles.">Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.</a> Investigative Ophthalmology &amp; Visual Science. 55 (5), 2893-2902 (2014).</li><li>Kur, J., Bankhead, P., Scholfield, C. 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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=TRPV2+Channels+Contribute+to+Stretch-Activated+Cation+Currents+and+Myogenic+Constriction+in+Retinal+Arterioles.">TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles.</a> Investigative Ophthalmology &amp; Visual Science. 57 (13), 5637-5647 (2016).</li><li>Guidoboni, G., 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=Intraocular+Pressure,+Blood+Pressure,+and+Retinal+Blood+Flow+Autoregulation:+A+Mathematical+Model+to+Clarify+Their+Relationship+and+Clinical+Relevance.">Intraocular Pressure, Blood Pressure, and Retinal Blood Flow Autoregulation: A Mathematical Model to Clarify Their Relationship and Clinical Relevance.</a> Investigative Ophthalmology &amp; Visual Science. 55 (7), 4105-4118 (2014).</li><li>Scholfield, C. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> Journal of Physiology. 532 (3), 609-623 (2001).</li><li>Tumelty, J., 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=Endothelin+1+stimulates+Ca2+-sparks+and+oscillations+in+retinal+arteriolar+myocytes+via+IP3R+and+RyR-dependent+Ca2++release.">Endothelin 1 stimulates Ca2+-sparks and oscillations in retinal arteriolar myocytes via IP3R and RyR-dependent Ca2+ release.</a> Investigative Ophthalmology &amp; Visual Science. 52 (6), 3874-3879 (2011).</li><li>Huynh, K. 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=Structural+insight+into+the+assembly+of+TRPV+channels.">Structural insight into the assembly of TRPV channels.</a> Structure. 22 (2), 260-268 (2014).</li><li>McGahon, M. K., Dawicki, J. M., Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A-type+potassium+current+in+retinal+arteriolar+smooth+muscle+cells.">A-type potassium current in retinal arteriolar smooth muscle cells.</a> Investigative Ophthalmology &amp; Visual Science. 46 (9), 3281-3287 (2005).</li><li>McGahon, M. K., Zhang, X., Scholfield, C. N., Curtis, T. M., McGeown, J. 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V., 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=Basement+membrane+abnormalities+in+human+eyes+with+diabetic+retinopathy.">Basement membrane abnormalities in human eyes with diabetic retinopathy.</a> Journal of Histochemistry and Cytochemistry. 44, 1469-1479 (1996).</li><li>Roy, S., Maiello, M., Lorenzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+basement+membrane+collagen+in+human+diabetic+retinopathy.">Increased expression of basement membrane collagen in human diabetic retinopathy.</a> Journal of Clinical Investigation. 93, 438-442 (1994).</li><li>Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+physiology+of+retinal+and+choroidal+arteriolar+smooth+muscle+cells.">Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.</a> Microcirculation. 14 (1), 11-24 (2007).</li><li>Hinds, K., Monaghan, K. P., Fr&#248;lund, B., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+control+of+arteriolar+diameter+in+the+rat+retina.">GABAergic control of arteriolar diameter in the rat retina.</a> Investigative Ophthalmology &amp; Visual Science. 54 (10), 6798-6805 (2013).</li><li>Torring, M. S., Aalkjaer, C., Bek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constriction+of+porcine+retinal+arterioles+induced+by+endothelin-1+and+the+thromboxane+analogue+U46619+in+vitro+decreases+with+increasing+vascular+branching+level.">Constriction of porcine retinal arterioles induced by endothelin-1 and the thromboxane analogue U46619 in vitro decreases with increasing vascular branching level.</a> Acta Ophthalmologica. 92 (3), 232-237 (2014).</li><li>P, S. k. o. v. . J. e. n. s. e. n. ., S, M. e. t. z. . M. a. r. i. e. n. d. a. l. . 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Dr. Joanna Kur is now an employee of Medtronic (Twin-Cities, Minnesota, USA), her present work does not conflict with that presented here.

The protocols described above require practice but should be achievable with minimal troubleshooting. On an average day we would obtain 6 - 8 usable arterioles from the isolation and achieve 3 - 4 successful experiments. If problems are encountered, however, there are some steps that can be taken to help improve success rates. Occasionally we have found, particularly when using younger rats (&#60; 8 weeks), that the yield of arterioles can be low. To circumvent this problem, we would suggest centrifuging the retinal tissue (10 - 30 s at 500 x g) between each of the trituration steps in steps 1.12 - 1.14. This often helps to improve the yield of vessels but will also increase the amount of cell debris within the preparation.
When cannulating vessels for pressure myography studies it is important to check the arterioles carefully for any side-branches that may have been cleaved close to the bifurcation site. This represents a common cause of leakage and loss of pressure during experimentation. The main issue that can arise with the [Ca2+]i protocols is the level of dye loading. Poor dye loading leads to low signal-to-noise ratios, while overloading results in disruption of normal Ca2+ homeostasis. To reduce the likelihood of either of these issues, a small aliquot of retinal homogenate can be removed, and isolated vessels checked periodically during the loading protocol. When undertaking patch-clamp experiments, the success rate of gigaseal formation is highly dependent on how well the basal lamina has been digested. When initially testing this protocol or using new lots of enzymes it may be necessary to adjust the concentration/duration of enzyme digestion. Carefully monitoring the separation of the endothelial and smooth muscle layers will ensure sufficient digestion to remove enough basal laminal to gain access. Careful monitoring is also necessary to avoid over-digestion, which can manifest as the vessel begins to constrict. If this occurs, the smooth muscle cells are often too fragile for patch clamp recording. When applying enzymes, it is important to include DNAse I to ensure removal of strands of DNA liberated from damaged cells during the isolation process. DNA fragments are sticky and cause the forceps to adhere to the arteriole during the final stages of the cleaning process (step 4.4), often resulting in vessel loss. Cleaning of vessels is technically difficult and is best performed at high magnification (20X) with gentle sweeping motions of closed forceps of the finest possible tip diameter. Between sweeps, clean the forceps with lab roll.
As highlighted earlier, a key motivation behind the development of the protocols outlined in this manuscript was to better understand why blood flow is disrupted during retinal vascular diseases. Most of our work to date has focused on diabetic eye disease28,33. Arterioles can be isolated from the retinas of experimental rodent models of diabetes using the methods described in section 1. When experimenting on isolated retinal arterioles from diabetic animals, it is important to try to closely replicate the hyperglycemic conditions experienced by the vessels in vivo. For this reason, we would normally raise the D-glucose levels in our isolation and experimental solutions to 25 mM. Thickening of vascular basement membranes is a well-recognized phenomenon in retinal vessels during diabetes34,35. When using animals with prolonged diabetes (&#62; 1 months disease duration), increased enzyme concentrations or digestion times are often needed to enable the application of patch-clamp recording methods.
An important limitation of using ex vivo isolated retinal arterioles to study retinal vascular physiology and pathophysiology is the loss of the surrounding retinal neuropile. Although removal of the retinal glial and neuronal cells enables easy access to the retinal vascular smooth muscle cells for cell physiology studies, the response of the vessels to vasoactive mediators can change dramatically in the absence and presence of retinal tissue. The actions of adenosine tri-phosphate (ATP), for example, provide a good illustration of this point. In isolated rat retinal arterioles, addition of ATP triggers a robust constriction of the vessels36, while in the presence of an intact neuropile, the vessels dilate37. Therefore, wherever possible, we usually try to validate key findings from our isolated arteriole preparations using ex vivo retinal whole-mounts and in vivo measurements of vessel diameter and blood flow16,37. Of note, new methods have recently emerged for studying small arterioles and capillaries in whole perfused porcine retinas ex vivo38,39. Such preparations are likely to greatly improve our understanding of how the retinal neuropile regulates retinal arteriolar and capillary tone and how changes in retinal haemodynamics modulate neuronal activity in the retina.
Although the procedures described in this paper are focused on the use of isolated rat retinal arterioles for understanding arteriolar smooth muscle cell physiology, we are currently developing protocols to also enable the study of endothelial cell function in these vessels. In preliminary work, we have been successful in modifying the enzymatic digestion of retinal arteriolar segments to yield viable endothelial cell tubes that are amenable to Ca2+ imaging and patchclamp recording studies. Cannulation of the isolated arterioles at both ends, enabling intraluminal delivery of drugs, could in the future also enable endothelium-dependent vasodilatory responses to be investigated in these vessels.

Understanding how blood flow is controlled in the retina is an important goal, since abnormal blood flow has been implicated in the pathogenesis of a variety of sight-threatening retinal diseases1,2,3,4. The retinal circulation, which supplies the inner retinal neurons and glial cells, has an end artery arrangement, with all of the blood from the retinal arteries and arterioles passing through the capillaries to the retinal venules and finally veins5. Blood flow in the retina is regulated by the tone of the retinal arteries and arterioles as well as the contractile activity of the pericytes located on the walls of the retinal capillaries and post-capillary venules6,7,8. The control of retinal vascular tone is complex and is modulated by a range of inputs from the circulatory system and surrounding retinal tissue, including blood gases, circulating molecules and hormones, and vasoactive substances released from the retinal vascular endothelium and macroglia9,10,11. The retinal arterioles are the small arterial branches of the retina and are composed of a single layer of vascular smooth muscle cells and an inner lining of longitudinally arranged endothelial cells12,13,14. These vessels form the main site of vascular resistance within the retinal circulation and therefore play an important role in the local control of retinal blood flow. Retinal arterioles regulate capillary blood flow in the retina by dilating or constricting their luminal diameter, mediated by changes in vascular smooth muscle contractility10,15,16. Understanding the molecular mechanisms through which retinal arterioles regulate retinal perfusion therefore requires preparations where the arteriolar smooth muscle cells can be accessed and studied in conditions as close to physiological as possible.
Ex vivo preparations of isolated retinal blood vessels provide access to the vascular smooth muscle cells, whilst still retaining their functionality and interconnectivity with the underlying endothelium. Most studies to date using isolated vessels have focused on large bovine or porcine arterial vessels (60 - 150 &#181;m). These can be mounted in commercially available wire or pressure myograph systems to enable pharmacological interrogation of vascular smooth muscle cell contractile mechanisms17,18. Such preparations have greatly contributed to our knowledge of retinal vascular physiology under normal conditions. Few studies have used retinal arterioles isolated from small laboratory animals as their smaller diameter (~ 8 - 45 &#181;m) prevents their use in conventional myography systems19,20,21,22. An important advantage, however, of using vessels from small laboratory animals is the wide availability of genetically modified, transgenic and retinal disease models. Small laboratory animals are also more amenable for in vivo intervention studies.
Here we describe straightforward protocols for isolating and cannulating rat retinal arterioles for pressure myography experiments. Ca2+ imaging and electrophysiology protocols using these vessels are also detailed. These can provide further insights into the regulation of vascular smooth muscle contractility and blood flow in the retina.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beakers</td>
			<td>Fisherbrand</td>
			<td>15409083</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Curved Scissors</td>
			<td>Fisher Scientific</td>
			<td>50-109-3542</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Disposable plastic pipette/ transfer</td>
			<td>Sarstedt</td>
			<td>86.1174</td>
			<td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Brunel Microscopes LTD</td>
			<td></td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>World Precision Instruments</td>
			<td>Dumont #5 14095</td>
			<td>Any equivalent fine forceps</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Fisherbrand</td>
			<td>11546963</td>
			<td>Fire polished to reduce friction but not enough to narrow the tip</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sigma-Aldrich</td>
			<td>P7741</td>
			<td>Or any equilavent 10cm product</td>
		</tr>
		<tr>
			<td>Pipette teat</td>
			<td>Fisherbrand</td>
			<td>12426180</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Purified water supply</td>
			<td>Merek</td>
			<td>Milli-Q Integral Water Purification System</td>
			<td>Or any equilavent system</td>
		</tr>
		<tr>
			<td>Round bottomed test tube</td>
			<td>Fisherbrand</td>
			<td>14-958-10 B</td>
			<td>5 mL; any equilavent product</td>
		</tr>
		<tr>
			<td>Seratted forceps</td>
			<td>Fisher Scientific</td>
			<td>17-467-230</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Single edge blades</td>
			<td>Agar Scientific</td>
			<td>T585</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Dow Corning</td>
			<td>184</td>
			<td>Or any pliable surface&#160;</td>
		</tr>
		<tr>
			<td>Testtube rack</td>
			<td>Fisherbrand Derlin</td>
			<td>10257963</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pressure myography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-axis mechanical manipulator</td>
			<td>Scientifica</td>
			<td>LBM-7&#160;</td>
			<td>x2 (or any equilavent product)</td>
		</tr>
		<tr>
			<td>3-way taps</td>
			<td>Cole-Parmer</td>
			<td>UY-30600-02</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Technical Manufacturing Corporation&#160;</td>
			<td>Clean Bench</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Analysis software&#160;</td>
			<td>Image J</td>
			<td>https://downloads.imagej.net/fiji/</td>
			<td>Free imaging software</td>
		</tr>
		<tr>
			<td>Analysis plugin</td>
			<td>Myotraker&#160;</td>
			<td>https://doi-org.remotexs.ntu.edu.sg/10.1371/journal.pone.0091791.s002</td>
			<td>Custom plugin for imageJ Freely available for download</td>
		</tr>
		<tr>
			<td>Cannulation pipette glass</td>
			<td>World Percision instruments</td>
			<td>TW150F-4</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Optiplex 7010</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>RS</td>
			<td>206-3722</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Fluo-4-AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>Make 1 mM stock in DMSO</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>World Precision Instruments</td>
			<td>Dumont #7 14097</td>
			<td>Any equivalent fine forceps</td>
		</tr>
		<tr>
			<td>Fura-2-AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F1221</td>
			<td>Make 1 mM stock in DMSO</td>
		</tr>
		<tr>
			<td>Glass bottomed recording chamber</td>
			<td>Warner Instruments</td>
			<td>64-0759</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Helper pipette glass</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>In-line heater</td>
			<td>Custom made</td>
			<td></td>
			<td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclypse TE 300</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Manometer</td>
			<td>Riester</td>
			<td>LF1459</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter instruments</td>
			<td>P97</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microforge +&#160; Olympus CX 31 microscope</td>
			<td>Glassworks</td>
			<td>Fine Point F-550</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter instruments</td>
			<td>MP-285</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Multi-channel delivery manifold&#160;</td>
			<td>Automate Scientific</td>
			<td>Perfusion Pencil</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipette filler&#160;</td>
			<td>BD Plastipak</td>
			<td>1mL</td>
			<td>Syringe heated and pulled to internal diameter of pipette</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Suction pump</td>
			<td>Interpet</td>
			<td>AP1</td>
			<td>Converted aeration pump by reversing bellows</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>20mL Luer-lok</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Advent</td>
			<td>W558818</td>
			<td>75&#956;m diameter</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>miscellaneous sizes</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>USB camera</td>
			<td>&#160;Logitech&#160;</td>
			<td>HD Pro Webcam C920</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Video capture software</td>
			<td>Hypercam</td>
			<td>version 2.28.01</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>Sub Aqua 12 Plus</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Nikon</td>
			<td>20x/0.40 WD 3.8, &#8734;/0.17</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Nikon</td>
			<td>4x/0.10, WD 30, &#8734;/-</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>World Percision Instruments</td>
			<td>14012</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Patch clamping</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-way taps</td>
			<td>Cole-Parmer</td>
			<td>UY-30600-02</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>3-axis mechanical manipulator</td>
			<td>Scientifica</td>
			<td>LBM-7&#160;</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Technical Manufacturing Corporation&#160;</td>
			<td>Clean Bench</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>3 mg disolved daily in 50 &#956;L of DMSO (sonicate to dissolve)</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Axopatch 200a</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Analogue digital converter</td>
			<td>Axon</td>
			<td>Digidata 1440A</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Collagenase Type 1A</td>
			<td>Sigma-Aldrich</td>
			<td>C9891&#160;</td>
			<td>0.1mg/mL in LCH</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Optiplex 7010</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>RS</td>
			<td>206-3722</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Millipore</td>
			<td>260913</td>
			<td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&#160;</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Custom made</td>
			<td></td>
			<td>Any equilavent commerical product</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>No. 7</td>
			<td>Any superfine forcep</td>
		</tr>
		<tr>
			<td>Glass bottomed recording chamber</td>
			<td>Warner Instruments</td>
			<td>64-0759</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>Molecular Devices</td>
			<td>CV203BU</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>In-line heater</td>
			<td>Custom made</td>
			<td></td>
			<td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclypse TE 300</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter instruments</td>
			<td>P97</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microforge +&#160; Olympus CX 31 microscope</td>
			<td>Glassworks</td>
			<td>Fine Point F-550</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter instruments</td>
			<td>MP-285</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Multi-channel delivery manifold&#160;</td>
			<td>Automate Scientific</td>
			<td>Perfusion Pencil</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Patching software</td>
			<td>Molecular Devices</td>
			<td>Pclamp v10.2</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipette filler&#160;</td>
			<td>BD Plastipak</td>
			<td>1mL</td>
			<td>Syringe heated and pulled to internal diameter of pipette</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Protease Type XIV</td>
			<td>Sigma-Aldrich</td>
			<td>P5147&#160;</td>
			<td>0.01mg/mL in LCH</td>
		</tr>
		<tr>
			<td>Single channel pipette glass</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Suction pump</td>
			<td>Interpet</td>
			<td>AP1</td>
			<td>Converted aeration pump by reversing bellows</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>20mL Luer-lok</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Advent</td>
			<td>W557418</td>
			<td>50&#956;m diameter</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>miscellaneous sizes</td>
			<td>Any equilavent product to fit</td>
		</tr>
		<tr>
			<td>USB camera</td>
			<td>&#160;Logitech&#160;</td>
			<td>HD Pro Webcam C920</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>Sub Aqua 12 Plus</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Whole cell pipette glass</td>
			<td>Warner Instruments</td>
			<td>GC150TF-7.5</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Nikon</td>
			<td>20x/0.40 WD 3.8, &#8734;/0.17</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Nikon</td>
			<td>4x/0.10, WD 30, &#8734;/-</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x40 lens</td>
			<td>Nikon</td>
			<td>40x/0.55, WD 2.1, &#8734;/1.2</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>World Percision Instruments</td>
			<td>14012</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Ca2+ imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-way taps</td>
			<td>Cole-Parmer</td>
			<td>UY-30600-02</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>3-axis mechanical manipulator</td>
			<td>Scientifica</td>
			<td>LBM-7&#160;</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Acquisition software&#160;</td>
			<td>Cairn Research Ltd.</td>
			<td>Acquisition Engine V1.1.5</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Technical Manufacturing Corporation&#160;</td>
			<td>Clean Bench</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Analysis software for confocal imaging</td>
			<td>Image J</td>
			<td>https://downloads.imagej.net/fiji/</td>
			<td>Free imaging software</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Optiplex 7010</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica Geosystems</td>
			<td>SP5&#160;</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>RS</td>
			<td>206-3722</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>No. 7</td>
			<td>Any superfine forcep</td>
		</tr>
		<tr>
			<td>Glass bottomed recording chamber</td>
			<td>Warner Instruments</td>
			<td>64-0759</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>In-line heater</td>
			<td>Custom made</td>
			<td></td>
			<td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE2000</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>Monochromator&#160;</td>
			<td>Cairn Research Ltd.</td>
			<td>Optoscan</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>Multi-channel delivery manifold&#160;</td>
			<td>Automate Scientific</td>
			<td>Perfusion Pencil</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipette filler&#160;</td>
			<td>BD Plastipak</td>
			<td>1mL</td>
			<td>Syringe heated and pulled to internal diameter of pipette</td>
		</tr>
		<tr>
			<td>Software for confocal microscope</td>
			<td>Leica Geosystems</td>
			<td>LAS-AF version 3.3.</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>Suction pump</td>
			<td>Interpet</td>
			<td>AP1</td>
			<td>Converted aeration pump by reversing bellows</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>20mL Luer-lok</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Advent</td>
			<td>W557418</td>
			<td>50&#956;m diameter</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>miscellaneous sizes</td>
			<td>Any equilavent product to fit</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>Sub Aqua 12 Plus</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x100 lens</td>
			<td>Nikon</td>
			<td>x100 N.A. 1.3 oil</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Nikon</td>
			<td>20x/0.40 WD 3.8, &#8734;/0.17</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Leica Geosystems</td>
			<td>HCX PL FLUOTAR, 20x/0.50, &#8734;/0.17/D</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Nikon</td>
			<td>4x/0.10, WD 30, &#8734;/-</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Leica Geosystems</td>
			<td>C PLAN, 4x/0.10, &#8734;/-/&#10013;</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>x63 lens</td>
			<td>Leica Geosystems</td>
			<td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &#8734;/0.17/E</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>World Percision Instruments</td>
			<td>14012</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td></td>
			<td></td>
			<td>Specifications and settings for fabrication of pipettes for experimentation</td>
		</tr>
		<tr>
			<td>Helper pipette</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Inner diameter: 0.86 mm 
			Ramp: 261 
			Pressure: 300 
			Heat: 280 
			Pull : 0 
			Velocity: 56 
			Time: 250 
			No of cycles: 4 
			Tip diameter: 0.5-2 &#956;m 
			Polishing: yes 
			Final Resistance: &#62;10 M&#937;</td>
		</tr>
		<tr>
			<td>Cannulation pipette</td>
			<td>World Percision instruments</td>
			<td>TW150F-4</td>
			<td>Inner diameter: 1.17 mm 
			Ramp: 290 
			Pressure: 300 
			Heat: 280 
			Pull : 0 
			Velocity: 58 
			Time: 150 
			No of cycles: 4 
			Tip diameter: 3-10 &#956;m 
			Polishing: no 
			Final Resistance: &#60;1 M&#937;</td>
		</tr>
		<tr>
			<td>On-cell patching</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Inner diameter: 0.86 mm 
			Ramp: 261 
			Pressure: 300 
			Heat: 280 
			Pull : 0 
			Velocity: 56 
			Time: 250 
			No of cycles: 4 
			Tip diameter: 0.5-2 &#956;m 
			Polishing: yes 
			Final Resistance: &#62;5 M&#937;</td>
		</tr>
		<tr>
			<td>Whole-cell patching</td>
			<td>Warner Instruments</td>
			<td>GC150TF-7.5</td>
			<td>Inner diameter: 1.17 mm 
			Ramp: 296 
			Pressure: 200 
			Heat: 287 
			Pull : 0 
			Velocity: 50 
			Time: 250 
			No of cycles: 4 
			Tip diameter: 2-3 &#956;m 
			Polishing: helpful but not necessary 
			Final Resistance: 1-2 M&#937;</td>
		</tr>
	</tbody>
</table>

isolation of retinal arterioles for ex vivo cell physiology studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

isolation-of-retinal-arterioles-for-ex-vivo-cell-physiology-studies

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K Views.  Queen's University of Belfast. This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies. 

Video: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...