Name: scalable, flexible, and cost-effective seedling grafting
Uploaded: 2023-01-06T13:20:00
Description: Watch this Scientific Journal Video about Scalable, Flexible, and Cost-Effective Seedling Grafting at JoVE.com

Thanks to Javier Brumos for initial training and guidance in grafting Arabidopsis seedlings.

1. Device preparation
<ol>
	<li>Make the silicone grafting device by casting silicone elastomer solution in a square Petri dish (100 mm x 100 mm). Prepare 15 mL of the elastomer solution, following the manufacturer&#39;s guidelines. 
	NOTE: Silicone elastomer kits typically include a silicone-based liquid and a curing agent, that when mixed together allow the silicone to solidify.</li>
	<li>Prepare the square Petri dish by laying four straight pieces of 29 G wire in the square Petri dish, equidist...

<ol>
	<li>Mudge, K., Janick, J., Scofield, S., Goldschmidt, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+history+of+grafting.">A history of grafting.</a> Horticultural Reviews. 35, 437-493 (2009).</li><li>Holbrook, N. M., Shashidhar, V. R., James, R. A., Munns, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+control+in+tomato+with+ABA-deficient+roots:+Response+of+grafted+plants+to+soil+drying.">Stomatal control in tomato with ABA-deficient roots: Response of grafted plants to soil drying.</a> Journal of Experimental Botany. 53 (373), 1503-1514 (2002).</li><li>Notaguchi, M., Okamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+long-distance+signaling+via+plant+vascular+tissues.">Dynamics of long-distance signaling via plant vascular tissues.</a> Frontiers in Plant Science. 6, 161 (2015).</li><li>Ko, D., Helariutta, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoot-root+communication+in+flowering+plants.">Shoot-root communication in flowering plants.</a> Current Biology. 27 (17), 973-978 (2017).</li><li>Thomas, H. R., Frank, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+the+pieces:+uncovering+the+molecular+basis+for+long-distance+communication+through+plant+grafting.">Connecting the pieces: uncovering the molecular basis for long-distance communication through plant grafting.</a> New Phytologist. 223 (2), 582-589 (2019).</li><li>Takahashi, F., 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=A+small+peptide+modulates+stomatal+control+via+abscisic+acid+in+long-distance+signalling.">A small peptide modulates stomatal control via abscisic acid in long-distance signalling.</a> Nature. 556 (7700), 235-238 (2018).</li><li>Brumos, 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=Local+auxin+biosynthesis+is+a+key+regulator+of+plant+development.">Local auxin biosynthesis is a key regulator of plant development.</a> Developmental Cell. 47 (3), 306-318 (2018).</li><li>Corbesier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FT+protein+movement+contributes+to+long-distance+signaling+in+floral+induction+of+Arabidopsis.">FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis.</a> Science. 316 (5827), 1030-1033 (2007).</li><li>Yin, H., 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=Graft-union+development:+A+delicate+process+that+involves+cell-cell+communication+between+scion+and+stock+for+local+auxin+accumulation.">Graft-union development: A delicate process that involves cell-cell communication between scion and stock for local auxin accumulation.</a> Journal of Experimental Botany. 63 (11), 4219-4232 (2012).</li><li>Turnbull, C. G. N., Booker, J. P., Leyser, H. M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micrografting+techniques+for+testing+long-distance+signalling.">Micrografting techniques for testing long-distance signalling.</a> The Plant Journal. 32 (2), 255-262 (2002).</li><li>Marsch-Mart&#237;nez, 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=An+efficient+flat-surface+collar-free+grafting+method+for+Arabidopsis+thaliana+seedlings.">An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings.</a> Plant Methods. 9 (1), 14 (2013).</li><li>Tsutsui, H., 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=Micrografting+device+for+testing+systemic+signaling+in+Arabidopsis.">Micrografting device for testing systemic signaling in Arabidopsis.</a> The Plant Journal. 103 (2), 918-929 (2020).</li><li>Xia, C., 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=Elucidation+of+the+mechanisms+of+long-distance+mRNA+movement+in+a+Nicotiana+benthamiana/tomato+heterograft+system.">Elucidation of the mechanisms of long-distance mRNA movement in a Nicotiana benthamiana/tomato heterograft system.</a> Plant Physiology. 177 (2), 745-758 (2018).</li><li>Li, 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=Unidirectional+movement+of+small+RNAs+from+shoots+to+roots+in+interspecific+heterografts.">Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts.</a> Nature Plants. 7 (1), 50-59 (2021).</li><li>Ragni, L., Hardtke, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+but+thick+enough-the+Arabidopsis+hypocotyl+as+a+model+to+study+secondary+growth.">Small but thick enough-the Arabidopsis hypocotyl as a model to study secondary growth.</a> Physiologia Plantarum. 151 (2), 164-171 (2014).</li><li>Chen, I. -. 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=A+chemical+genetics+approach+reveals+a+role+of+brassinolide+and+cellulose+synthase+in+hypocotyl+elongation+of+etiolated+Arabidopsis+seedlings.">A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings.</a> Plant Science. 209, 46-57 (2013).</li><li>An, F., 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=Coordinated+regulation+of+apical+hook+development+by+gibberellins+and+ethylene+in+etiolated+Arabidopsis+seedlings.">Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings.</a> Cell Research. 22 (5), 915-927 (2012).</li><li>Vandenbussche, F., 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=Ethylene-induced+Arabidopsis+hypocotyl+elongation+is+dependent+on+but+not+mediated+by+gibberellins.">Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins.</a> Journal of Experimental Botany. 58 (15-16), 4269-4281 (2007).</li><li>Vandenbussche, F., 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+Arabidopsis+mutant+alh1+illustrates+a+cross+talk+between+ethylene+and+auxin.">The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin.</a> Plant Physiology. 131 (3), 1228-1238 (2003).</li><li>Deslauriers, S. D., Larsen, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FERONIA+is+a+key+modulator+of+brassinosteroid+and+ethylene+responsiveness+in+arabidopsis+hypocotyls.">FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in arabidopsis hypocotyls.</a> Molecular Plant. 3 (3), 626-640 (2010).</li></ol>

Summary and significance 
Formation of a graft union is crucial for successful grafting, which requires direct and undisturbed contact between the rootstock and scion. The miniature size and fragility of seedlings of small plants such as Arabidopsis makes it technically challenging to meet this requirement. One technique developed in early Arabidopsis seedling grafting methods was to insert both the scion and the rootstock into a short silicone tubing collar to support the graft ju...

Grafting is an ancient horticultural technique that became an established agricultural practice by 500 BCE1. Grafting different varieties of crop plants to improve yields was the first use of this technique, and continues to be used for this purpose today. In the past decade, grafting has attracted an increasing amount of attention as a tool for molecular biologists to study long-distance signaling in plants2,3,4,5. While grafting adult plants is relatively easy, grafting plants soon after germination is challenging. Despite this, it is sometimes required to assess the effects of long-distance signaling in processes such as plant development, environmental responses, and flowering6,7,8.
Arabidopsis thaliana has been established as the model organism in plant biology for many reasons, including its relatively small size, rendering it easy to grow inside a lab. However, the small size and fragility of Arabidopsis seedlings makes grafting young seedlings very challenging. In many cases, extensive hands-on training is required to successfully obtain seedling grafts. There have been many methodological improvements over the years that have identified ideal growing conditions and new techniques to increase the success rate of seedling grafting9,10,11. The most recent tool introduced was an Arabidopsis seedling grafting chip, that allows even inexperienced users to achieve acceptable levels of grafting success12. While this advance has significantly lowered the technical barrier of seedling grafting, the chip device is expensive, and the number of grafts that can be conducted in parallel quickly becomes cost-prohibitive.
Additionally, this device can only be used for Arabidopsis seedlings that have hypocotyl dimensions that are similar to wild-type seedlings. While Arabidopsis is the keystone species in the world of plant molecular genetics, recent work has been done in other species using seedling grafting. Examples include the grafting of soybean and the common bean, tobacco to tomato, and canola to Arabidopsis, and subsequently sampling both tissues for small RNAs13,14. Therefore, a grafting method that is accessible to most laboratories and can be easily adapted to a wide range of plant species without any major technique changes is highly desirable.
This protocol details a method that employs in-house production of a simple grafting device that allows for the full customization of grafting channel diameter and length to accommodate any seedling morphology across most plant species. The production of these devices is very affordable and highly scalable, as the only components needed are silicone elastomer, wiring or tubing of the correct size, a high precision blade, and a container to serve as a mold. Following the grafting protocol detailed here, users can achieve successful grafting rates of 45% (n = 105), comparable with previously reported grafting results10,12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR International Inc</td>
			<td>10026-076</td>
			<td></td>
		</tr>
		<tr>
			<td>ACETONE (HPLC &#38; ACS Certified Solvent) 4 L</td>
			<td>VWR</td>
			<td>BJAH010-4</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoAgar</td>
			<td>Sigma</td>
			<td>A1296-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow SYLGARD 184 Silicone Encapsulant Clear 0.5 kg Kit</td>
			<td>Dow</td>
			<td>2646340</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sucrose (Molecular Biology), 1 kg</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Flex-Tube Tubes (1.5 mL), pack of 500</td>
			<td>Fisher Scientific</td>
			<td>20901-551 / 05-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision #4 Style Scalpel Handle</td>
			<td>Fisher Scientific</td>
			<td>12-000-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Lead-Free Autoclave Tape</td>
			<td>Fisher Scientific</td>
			<td>15-901-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand square petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Zoom 2000 Stereo Microscope</td>
			<td>Microscope Central</td>
			<td>L-Z2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore Tape</td>
			<td>3M</td>
			<td>B0082A9FEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Medium</td>
			<td>Sigma</td>
			<td>M5519-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Genesee Scientific</td>
			<td>16-101</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>VWR International Inc</td>
			<td>AA13451-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Redi-earth Plug and Seedling Mix</td>
			<td>Sun Gro Horticulture</td>
			<td>SUN239274728CFLP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotts Osmocote Plus</td>
			<td>Hummert International</td>
			<td>7630600</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design No. 22 Carbon Scalpel Blade</td>
			<td>Fisher Scientific</td>
			<td>22-079-697</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20, 500 mL</td>
			<td>Fisher Scientific</td>
			<td>BP337500</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEZER DUMONT STYL55 DUMOXEL POLS 110 MM</td>
			<td>VWR</td>
			<td>102091-580</td>
			<td></td>
		</tr>
	</tbody>
</table>

scalable, flexible, and cost-effective seedling grafting

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage seedlings of the small model plant, Arabidopsis thaliana, is technically challenging and time consuming due to the size and fragility of its seedlings. A growing collection of published methods describe this technique with varying success rates, difficulty, and associated costs. This paper describes a simple procedure to make an in-house reusable grafting device using silicone elastomer mix, and how to use this device for seedling grafting. At the time of this publication, each reusable grafting device costs only $0.47 in consumable materials to produce. Using this method, beginners can have their first successfully grafted seedlings in less than 3 weeks from start to finish. This highly accessible procedure will allow plant molecular genetics labs to establish seedling grafting as a normal part of their experimental process. Due to the full control users have in the creation and design of these grafting devices, this technique could be easily adjusted for use in larger plants, such as tomato or tobacco, if desired.

Various aspects of the grafting strip&#39;s design were tested to identify the optimal grafting conditions that required the least amount of technical skill (Table 1). All grafting trials were completed on 0.5% sucrose MS medium, which has been previously reported to be an ideal grafting medium11,12.
Optimal seedling growth cannot be achieved with on-strip germination 
In the first iteration of th...

Watch this Scientific Journal Video about Scalable, Flexible, and Cost-Effective Seedling Grafting at JoVE.com

Scalable, Flexible, and Cost-Effective Seedling Grafting

This protocol describes a robust seedling grafting method that requires no prior experience or training and can be executed at a very low cost using materials easily accessible in most molecular biology labs.

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage ...

Early stage seeding grafting is a powerful tool for setting long-distance signaling in plants. We hope that this method will aid in the successful adoption of seeding grafting without hands-on training. Begin by preparing the square Petri dish, measuring 100x100 millimeters for casting the silicone elastomer.Take 29 gauge wire in twist ties after removing the outer paper coating on the wire with acetone. And fully straighten the wire by rolling it on a hard, uniform surface with a heavy flat object, like a metal tube rack. Then, lay four pieces of straightened wires equidistant from one another in the square Petri dish, ensuring the wires lie flat on the bottom of the dish.Pour the mixed silicon elastomer solution on top of the wires in the Petri dish and place the cover on the dish. After allowing the silicone to cure for 24 to 48 hours at room temperature, remove the silicone sheet from the Petri dish using clean forceps and move it to a clean, flat surface. Remove the wires from the silicone sheet.To ensure the channels are open on one side, use fine-point forceps to clear the thin layer of silicone blocking the open side of the channels. Cut the silicone sheet perpendicular to the channels into three-millimeter strips using clean scissors. Place each strip in an aluminum foil envelope and seal it with autoclave tape.Autoclave the strips at 121 degrees Celsius for at least 30 minutes before storing them until required. To sterilize and vernalize the seeds, take one milliliter of 50%bleach solution containing 0.1%Tween 20 in a 1.5-milliliter microcentrifuge tube and suspend up to 100 arabidopsis seeds in it. Incubate the seed suspension at room temperature for five to 10 minutes.Post-incubation, aspirate or pipette out the bleach solution under sterile conditions. Rinse the seeds with one milliliter of sterilized distilled water. Be sure to invert the tubes adequately while rinsing the seeds.To remove any bleach solution left at the top of the tube, repeat the rinsing four times. Leave approximately 250 microliters of water with the seeds in the tube, and store the seeds in the dark at four degrees Celsius for three days. Next, before plating the seeds, place the sterile strips on the surface of the prepared MS plate to guide the seed alignment with the channels on the strips.Then, working under sterile conditions, use a 20-microliter pipette tip to aspirate and transfer the appropriate number of prepared seeds onto the plate. Once the seeds are plated, remove the sterile strips. Cover the plate.And seal one of the sides of the plate parallel to the rows of seeds with parafilm. Wrap breathable tape on top of the parafilm and around all the other edges of the plate to allow for air exchange during incubation. Carefully orient two plates vertically with their parafilm-sealed sides facing down.Separate them at the bottom by horizontally placing a 15-milliliter centrifuge tube in between, while securing them together on top with a rubber band. Ensure the plate surfaces form an angle of 100 to 110 with the benchtop surface. Store the plates in this orientation for 72 hours in total darkness at 21 degrees Celsius to allow seedling hypocotyls to grow about five millimeters in length.After which, remove the setup from the dark and keep it under a 16-hour light and eight-hour dark cycle for two to four days at the same temperature before grafting. Graft the seedlings between five and seven days after being plated. Place a grafting strip over the seedlings, fitting their hypocotyls into the channels.Gently position the seedling, so the root-hypocotyl junction remains at the bottom of the silicone strip to prepare the seedling for cutting. Working in a sterile hood with the aid of a dissection scope, prepare the scions for grafting. Using a fresh scalpel blade, make a straight, clean, perpendicular cut across the hypocotyl.Avoid pressing down and pushing the seedling into the agar. Next, remove the chute and keep the cut portion of the chute hydrated by ensuring contact with the media surface. Alternatively, the chute can be moved to a Petri dish filled with sterile distilled water.To prepare the rootstocks, carefully grasp the root within the space between the closed forceps and turn gently to pull it, leaving the cut section of the rootstocks in the middle of the strip. Then, gently pick up the desired chute using the fine-tipped forceps and insert it into the top of the the channel. Visually, confirm the contact between the scion and rootstock to obtain a successful graft.After all the grafts have been made, wrap the plates with parafilm and breathable tape, and set up the plates the same way as demonstrated before without disturbing the seedlings or silicone strips. Carefully move the plates to a growth chamber set at 26 degrees Celsius with a 16-hour light and eight-hour dark cycle. After seven to 10 days, evaluate the grafted seedlings under sterile conditions by using forceps to carefully peel off the silicone strip from one side and free the seedlings from the channels.Remove any a adventitious roots growing from the scion by cutting them with a fresh scalpel blade or crushing them with fine-tipped forceps. Once again, visually evaluate whether the rootstock has become firmly attached to the scion forming a successful graft. Move the successful grafts to seedling propagation soil and cover the soil with transparent plastic for a few days while the seedlings get established.Allow the plants to grow under the previously mentioned light and dark cycles at 21 degrees Celsius. Grafting trials to determine the optimal grafting protocol revealed an overall grafting success rate of 25%when the seeds germinated directly on the silicone strips with enclosed channels. Reorienting the seeds to keep the embryos'cotyledons and radical pointing downward during germination slightly increased the grafting efficiency to 33%Upon removing the silicone closing on the channels to allow contact between the seed and the growth medium, 85%of the seedlings germinated successfully.However, the grafting success rate remained at 31%indicating that optimal seedling growth could not be achieved with on-strip germination. Interestingly, the demonstrated protocol in which seedlings were cultivated vertically on the grafting plate before being inserted into the open channels in the strips yielded an overall grafting success rate to 48%during the first two trials. Considering the lower success rate of the third and the relatively small trial, which may be attributed to slight variations in grafting sites, the overall grafting success was found to be 45%This is comparable with the method where the seedlings were cultivated on horizontal plates, cut on a solid surface, and inserted into grafting strips.However, the demonstrated protocol is comparatively less time-consuming. Using this protocol, labs new to seedling grafting can rapidly create large numbers of grafting support devices while being able to customize the device to fit their specific needs easily.

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