The authors acknowledge financial support from CannabiVar Ltd. All plant material was generously provided by Professor Hinanit Koltai from the Volcani Center, Israel.

NOTE: The plant material used in this study consisted of four C. sativa ARO-Volcani strains (CS-11, CS-12, CS-13, and CS-14) that were grown in the Volcani Center, Israel, as described elsewhere11. Glandular capitate stalked trichomes were isolated from mature flowering inflorescences, and glandular capitate sessile trichomes were isolated from large fan leaves from mature non-flowering mother plants. All plant material was freshly picked and immediately stored at &#8722;80&#160;&#176;C.
CAUTION: Dry ice and LN are used throughout the protocol. These substances are extremely hazardous. The isolated trichomes can contain dry ice particles that can create dangerous gas pressure when inserted into sealed tubes; therefore, all caps should be needle-punctured. The use of protective goggles, appropriate lab wear, and gloves for handling at extremely low temperatures is highly advised.
1. Setup for the initial separation of trichomes from the plant material
<ol>
	<li>Crush a dry ice block into small fine flakes using a hammer and a hard flat object in a 5 L plastic container.</li>
	<li>Sieve the fine dry ice flakes (smaller than 5 mm) from the non-crushed dry ice with a large strainer (via pores smaller than 5 mm) into another 5 L plastic container. Pour approximately 200 cm3 (200 mL mark) of the dry ice flakes into an upright 1 L glass beaker.</li>
	<li>Add up to 10 g of frozen C. sativa inflorescences (from now on referred to as plant material) onto the first layer of crushed dry ice, and cover with an additional layer of 200 cm3 of finely crushed dry ice.</li>
	<li>Cover the opening of the 1 L glass beaker with two to three layers of 1 mm screen door mosquito net, and secure it to the outer sides of the glass cup with rubber bands (Figure 1).</li>
	<li>Pour LN&#160;into a large round-bottom stainless steel container (see Table of Materials), where the isolated trichomes will be collected.</li>
	<li>Insert a 350 &#181;m mesh in a flour sifter/sieve strainer to cover the flour sifter mesh from below (Figure 2). If a flour sifter with a detachable plastic ring is available, insert the 350 &#181;m mesh so that it is secured under the flour sifter&#39;s mesh. If not, secure the 350 &#181;m mesh to the circumference of the flour sifter with rubber bands.</li>
	<li>Place the flour sifter above the large round-bottom stainless steel container filled with LN (Figure 3). Ensure that the width of the container exceeds that of the flour sifter to minimize the loss of the sifted mass outside the round-bottom stainless steel container.</li>
</ol>
2. Separation of trichomes from the plant material
<ol>
	<li>Shake the 1 L glass with its opening pointing downward toward the flour sifter as if using a large salt shaker (Figure 4).</li>
	<li>Set aside the 1 L glass beaker every 2-3 min to allow the sifting of the crushed dry ice and plant material accumulated on the flour sieve.</li>
	<li>Sift the flour sieve horizontally to facilitate the passage of the plant material into the LN&#160;in the round-bottom stainless steel container below.</li>
	<li>Add LN to the stainless steel container and crushed dry ice to the 1 L glass beaker when their levels run low. Replenish the used plant material in the glass beaker with an additional 10 g of fresh plant material when the plant material on the flour sieve is depleted. The replenishment usually does not have to be repeated more than twice.</li>
	<li>Repeat steps 2.1-2.4 until a sufficient amount of enriched trichomes has been collected in the round-bottom stainless steel container. Generally, 20 g of fresh plant material is sufficient for RNA extraction and transcriptome analysis.</li>
	<li>Verify that there is a white powder-like substance (consisting of plant debris, glandular trichomes, and crushed dry ice) at the bottom of the stainless steel container submerged in the LN. A microscopic observation will help to determine if a sufficient amount of trichomes has been collected; however, this step might obstruct the protocol&#39;s flow. Usually, 10-20 g of initial plant material suffices for most isolations; however, there may be differences in the trichome density of the plants. 
	NOTE: From this point, the experiment can be paused and restarted later. If paused for a short time, adequate amounts of LN&#160;should be added so that the trichomes remain submerged. Alternatively, the trichomes can be collected and stored at &#8722;80 &#176;C for up to 3 months for later use, as described in step 5.</li>
</ol>
3. Separation of glandular capitate trichomes (stalked and sessile) from other trichome types, such as bulbous and cystolithic trichomes, and debris
<ol>
	<li>Add a small amount of LN&#160;to a clean small round-bottom stainless steel container.</li>
	<li>Fold a 40 cm x 40 cm micro-sieve with a 150 &#181;m mesh size twice to obtain a 20&#160;cm x 20&#160;cm fold, and open it. Ensure it resembles a cone-like shape (Figure 5).</li>
	<li>Secure the mesh cone to the edge of the round-bottom stainless steel container with one or two clothespins so that the opened part of the cone is upright and its pointed part is partly submerged in the LN.</li>
	<li>Gently pour the LN&#160;and the white powder-like substance from step 2.6 into the micro-sieve cone.</li>
	<li>Gently apply a wide brush to gather and transfer any remaining plant material from the first container into the 150 &#181;m mesh cone.</li>
	<li>Add more LN to the first container, and repeat the brushing motion until all the plant material is transferred to the micro-sieve cone.</li>
	<li>Carefully detach the clothespin from the lid of the container, and open the micro-sieve cone so that the trichomes are located in the middle of the opened micro-sieve. Hold all four corners of the micro-sieve together so that the middle part containing the trichomes remains submerged in the LN (Figure 6).</li>
	<li>While holding all four corners of the micro-sieve, gently dip and shake the micro-sieve in the LN in the steel container as if infusing a tea bag. Continue this dipping/shaking (vertical and horizontal) motion for 1 min. There is a tradeoff between the isolation quality and quantity. Longer dipping motions may result in larger amounts of trichomes, but their isolation grade may be poorer. Overall, 1 min is recommended as adequate for both quality and quantity. 
	NOTE: This sieving motion is the most critical part of the protocol and should be executed accordingly.</li>
	<li>Optional: Scoop the non-sifted plant debris and larger trichomes (still submerged in the LN) that are wrapped in the center of the micro-sieve into a 13 mL&#160;or 50 mL test tube, and store at &#8722;80 &#176;C for future use. 
	&#8203;CAUTION: To avoid the buildup of pressured gas (from the dry ice and LN residues), puncture a hole in the cap of the test tube with a sterilized needle. The cap can be replaced once the pressure has reduced, which is usually after 24 h.</li>
</ol>
4. Passing trichomes through micro-sieves of decreasing pore size
<ol>
	<li>Transfer the sifted trichomes submerged in the LN at the bottom of the small round-bottom stainless steel container sequentially through micro-sieves with decreasing pore sizes (105 &#181;m, 80 &#181;m, 65 &#181;m, and 50 &#181;m), in the same manner as presented in step 3.</li>
</ol>
5. Collection of the desired trichomes
<ol>
	<li>Remove the desired trichomes submerged in the LN and wrapped in the 50 &#181;m micro-sieve using a pre-chilled (in LN) spoon. Place them on a clean plate. 
	NOTE: In this final isolation stage, the desired trichomes are collected from the sieve.</li>
	<li>Quickly transfer the powder-like trichomes into a labeled pre-chilled 1.5 mL tube via a pre-chilled spatula or a scoopula inserted into the tube (Figure 7). Store the tubes immediately at &#8722;80 &#176;C for further studies. 
	&#8203;NOTE: A schematic workflow chart depicting the whole isolation protocol is given in Figure 8.</li>
</ol>
6. Observation and analysis of the purified trichomes
<ol>
	<li>Place a small amount (10 mg) of isolated trichomes on a microscope slide using a pre-cooled (via LN) spatula. Add a drop of water, and directly observe on a light microscope without staining. Evaluate the overall isolation and contamination degree using low (10x) and high (40x) magnifications. Enrichment is visually estimated by the relative amount of trichomes with respect to non-trichome tissue, as shown in Figure 9.</li>
	<li>Perform RNA extraction and quality analysis from 50 mg of isolated C. sativa glandular capitate stalked and sessile trichomes. 
	NOTE: A commercial extraction kit (see Table of Materials) employing a poly-A-based strategy of mRNA purification was used for the RNA extraction.
	<ol>
		<li>Resuspend 50 mg of the isolated trichomes in the lysis solution, vortex for 30 s, sonicate in an ice-cold ultrasonic cleaning unit at 35 kHz for 5 min, and extract the mRNA from the samples according to the manufacturer&#39;s protocol (see Table of Materials).</li>
		<li>To estimate the integrity of the RNA extracted from the trichomes, determine the RNA concentration, the total amount, and the RNA integrity number (RIN) values using a lab-on-a-chip system (see Table of Materials).</li>
		<li>Perform RNAseq analysis of the trichome fractions (optional). RNAseq analysis and bioinformatics analyses of sequenced reads can be performed by standard outsourcing services</li>
	</ol>
	</li>
</ol>

<ol><li>Schilmiller, A. L., Last, R. L., Pichersky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harnessing+plant+trichome+biochemistry+for+the+production+of+useful+compounds%5BTitle%5D)+AND+%22Plant+Journal+for+Cell+%26+Molecular+Biology%22%5BJournal%5D)">Harnessing plant trichome biochemistry for the production of useful compounds.</a> Plant Journal for Cell & Molecular Biology. 54 (4), 702-709 (2008).</li>
<li>Liu, Y., Jing, S. X., Luo, S. H., Li, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-volatile+natural+products+in+plant+glandular+trichomes%3A+chemistry%2C+biological+activities+and+biosynthesis%5BTitle%5D)+AND+%22Natural+Product+Reports%22%5BJournal%5D)">Non-volatile natural products in plant glandular trichomes: chemistry, biological activities and biosynthesis.</a> Natural Product Reports. 36 (4), 626-665 (2019).</li>
<li>Fairbairn, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+trichomes+and+glands+of+Cannabis+sativa+L%5BTitle%5D)+AND+%22UN+Bulletin+on+Narcotics%22%5BJournal%5D)">The trichomes and glands of Cannabis sativa L.</a> UN Bulletin on Narcotics. 23, 29-33 (1972).</li>
<li>Booth, J. K., Page, J. E., Bohlmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Terpene+synthases+from+Cannabis+sativa%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Terpene synthases from Cannabis sativa.</a> PLoS One. 12 (3), 0173911(2017).</li>
<li>Yerger, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+method+for+isolating+glandular+trichomes%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">A rapid method for isolating glandular trichomes.</a> Plant Physiology. 99 (1), 1-7 (1992).</li>
<li>Gershenzon, J., Maffei, M. M., Croteau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+and+histochemical+localization+of+monoterpene+biosynthesis+in+the+glandular+trichomes+of+spearmint+%28Mentha+spicata%29%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Biochemical and histochemical localization of monoterpene biosynthesis in the glandular trichomes of spearmint (Mentha spicata).</a> Plant Physiology. 89 (4), 1351-1357 (1989).</li>
<li>Gershenzon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+secretory+cells+from+plant+glandular+trichomes+and+their+use+in+biosynthetic+studies+of+monoterpenes+and+other+gland+products%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.</a> Analytical Biochemistry. 200 (1), 130-138 (1992).</li>
<li>Conneely, L. J., Mauleon, R., Mieog, J., Barkla, B. J., Kretzschmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+Cannabis+sativa+glandular+trichome+proteome%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Characterization of the Cannabis sativa glandular trichome proteome.</a> PLoS One. 16 (4), 0242633(2021).</li>
<li>Liu, Y., Zhu, P., Cai, S., Haughn, G., Page, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three+novel+transcription+factors+involved+in+cannabinoid+biosynthesis+in+Cannabis+sativa+L%5BTitle%5D)+AND+%22Plant+Molecular+Biology%22%5BJournal%5D)">Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.</a> Plant Molecular Biology. 106 (1-2), 49-65 (2021).</li>
<li>Slone, J. H., Kelsey, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+purification+of+glandular+secretory+cells+from+Artemisia+tridentata+%28ssp.+vaseyana%29+by+Percoll+density+gradient+centrifugation%5BTitle%5D)+AND+%22American+Journal+of+Botany%22%5BJournal%5D)">Isolation and purification of glandular secretory cells from Artemisia tridentata (ssp. vaseyana) by Percoll density gradient centrifugation.</a> American Journal of Botany. 72 (9), 1445-1451 (1985).</li>
<li>Namdar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Terpenoids+and+Phytocannabinoids+Co-Produced+in+Cannabis+Sativa+Strains+Show+Specific+Interaction+for+Cell+Cytotoxic+Activity%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Terpenoids and Phytocannabinoids Co-Produced in Cannabis Sativa Strains Show Specific Interaction for Cell Cytotoxic Activity.</a> Molecules. 24 (17), 3031(2019).</li>
<li>McDowell, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+functional+genomic+analysis+of+Solanum+glandular+trichome+types%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Comparative functional genomic analysis of Solanum glandular trichome types.</a> Plant Physiology. 155 (1), 524-539 (2011).</li>
<li>Bergau, N., Santos, A. N., Henning, A., Balcke, G. U., Tissier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autofluorescence+as+a+signal+to+sort+developing+glandular+trichomes+by+flow+cytometry%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Autofluorescence as a signal to sort developing glandular trichomes by flow cytometry.</a> Frontiers in Plant Science. 7, 949(2016).</li>
<li>Livingston, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cannabis+glandular+trichomes+alter+morphology+and+metabolite+content+during+flower+maturation%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Cannabis glandular trichomes alter morphology and metabolite content during flower maturation.</a> The Plant Journal. 101 (1), 37-56 (2019).</li>
<li>Turner, J. C., Hemphill, J. K., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gland+distribution+and+cannabinoid+content+in+clones+of+Cannabis+sativa+L%5BTitle%5D)+AND+%22American+Journal+of+Botany%22%5BJournal%5D)">Gland distribution and cannabinoid content in clones of Cannabis sativa L.</a> American Journal of Botany. 64 (6), 687-693 (1977).</li>
<li>Turner, J. C., Hemphill, J. K., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+determination+of+cannabinoids+in+individual+glandular+trichomes+of+Cannabis+sativa+L.+%28Cannabaceae%29%5BTitle%5D)+AND+%22American+Journal+of+Botany%22%5BJournal%5D)">Quantitative determination of cannabinoids in individual glandular trichomes of Cannabis sativa L. (Cannabaceae).</a> American Journal of Botany. 65 (10), 1103-1106 (1978).</li>
<li>Hammond, C. T., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphology+of+glandular+hairs+of+Cannabis+sativa+from+scanning+electron+microscopy%5BTitle%5D)+AND+%22American+Journal+of+Botany%22%5BJournal%5D)">Morphology of glandular hairs of Cannabis sativa from scanning electron microscopy.</a> American Journal of Botany. 60 (6), 524-528 (1973).</li>
<li>Marks, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+method+for+isolating+large+quantities+of+Arabidopsis+trichomes+for+transcriptome%2C+cell+wall%2C+and+other+types+of+analyses%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall, and other types of analyses.</a> The Plant Journal. 56 (3), 483-492 (2008).</li>
<li>Huebbers, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+advanced+method+for+the+release%2C+enrichment+and+purification+of+high-quality+Arabidopsis+thaliana+rosette+leaf+trichomes+enables+profound+insights+into+the+trichome+proteome%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome.</a> Plant Methods. 18 (1), 12(2022).</li>
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The authors have no conflicts of interest.

Compared to the currently available trichome isolation protocols, two main modifications are described in the present protocol. These include the detachment of the trichomes from the plant material using dry ice in the initial step and substituting LN for the commonly used liquid buffer medium. The first modification for C. sativa trichome purification is based on an earlier protocol that introduced the use of crushed dry ice to detach the trichomes from geranium pedicels5. While traditio...

Glandular trichomes are hair-like structures present in plants that contain many secondary metabolites1 and represent a valuable bank of novel biosynthetic genes and enzymes2. In Cannabis, the biosynthesis of the important secondary metabolites, cannabinoids3 and terpenes4, is localized in the trichomes. Considering the role of trichomes in determining the quality of Cannabis both for medicinal and recreational uses, the study of trichome gene expression is of interest. To characterize the expression of trichome-specific genes, the trichomes of interest must first be isolated. Trichome isolation protocols were first described as early as 19925, and their latest developments have been recently reviewed2. In general, protocols for extracting glandular trichomes for transcriptomic characterization can be divided into two distinct sequential steps. The first step involves a thorough physical separation of the trichomes from the plant tissue. This step can be performed by using dry ice5, glass beads with a commercial apparatus6,7, grinding the plant material against a mesh sieve8, or vortexing the plant tissue in an isolation buffer9. The second step involves a more refined separation of the trichomes of interest from the microscopic plant residue and/or other trichome types. This step can be executed using density gradient centrifugation8,10 or sieves of various sizes7,9. Due to the extreme sensitivity of RNA in processed tissues to degrading agents, these two sequential steps are usually conducted in the ice-cold isolation medium, often in the presence of protein inhibitors4.
Conventional trichome isolation protocols require, in addition to the ice-cold temperatures, large amounts of isolation medium to ensure an efficient extraction procedure. The combination of these components results in an arduous, time-consuming isolation process that hinders high throughput. Presenting a straightforward, user-friendly alternative trichome isolation protocol is, therefore, likely to be beneficial for various aspects associated with trichome characterization. The present paper aims to offer an alternative protocol for isolating stalked and sessile glandular capitate trichomes from Cannabis sativa by combining and integrating several elements from the conventional protocols. These elements include dry ice5, the passing of the trichomes through several micro-sieves with decreasing pore sizes7,9, and substituting liquid nitrogen (LN)&#160;for the isolation medium8.
The novelty of the present trichome isolation protocol, as compared to conventional protocols, presents in a number of ways. This protocol is convenient, as it does not require hazardous components. The procedure can be conducted in the lab with minimal precautions and facilitates high throughput. Substituting LN for the standard liquid isolation medium ensures the integrity of the trichomes throughout the isolation process, enabling subsequent transcriptomic analysis. Upon sublimation of the LN and dry ice, the isolated trichomes are left free of harmful residuals. Further, the propensity of LN to sublimate at room temperature allows its generous use throughout the protocol. In contrast, using large volumes of conventional isolation medium generates practical difficulties in its handling. Finally, the protocol decreases the separation of the disc cell from the remaining fragile head structure of the glandular trichome, enabling the retention of the headspace content.
This protocol is presented in a detailed step-by-step fashion designed to assist the technical practice of isolating C. sativa glandular capitate trichomes. The protocol provides a manageable workflow that results in isolated trichomes with a high concentration and purity that are appropriate for downstream molecular analysis.

<table><tbody><tr><td>Bioanalyzer RNA Pico 6000 chip</td><td>Agilent, Germany</td><td>Reorder number 5067-1513</td><td>Lab-on-a-chip system&amp;nbsp;</td></tr><tr><td>Transsonic-310</td><td>Elma, Germany</td><td>D-78224</td><td>Ultrasonic cleaning unit&amp;nbsp;</td></tr><tr><td>TruSeq RNA Sample Prep Kit v2</td><td>Illumina, USA</td><td>RS-122-2001</td><td>Sample preperation for RNA sequencing library</td></tr><tr><td>Spectrum Plant Total RNA Kit&amp;nbsp;</td><td>SIGMA-ALDRICH, USA</td><td>STRN50-1KT</td><td>Plant Total RNA Kit&amp;nbsp;</td></tr><tr><td>Nylon micro-sieve with a mesh size of 350 &amp;micro;m (40 x 40 cm or larger than the circumference of the flour sifter)</td><td>Sinun Tech, Israel</td><td>r0350n350210</td><td>Nylon&amp;nbsp;screen aperture</td></tr><tr><td>Nylon micro-sieve with mesh size of 150 &amp;micro;m (size of 30 x 30 cm)</td><td>Sinun Tech, Israel</td><td>r0150n360465</td><td>Nylon&amp;nbsp;screen aperture</td></tr><tr><td>Nylon micro-sieve with mesh size o 105 &amp;micro;m (size of 30 x 30 cm)</td><td>Sinun Tech, Israel</td><td>r0105n320718</td><td>Nylon&amp;nbsp;screen aperture</td></tr><tr><td>Nylon micro-sieve with mesh size o 80 &amp;micro;m (size of 30 x 30 cm)</td><td>Sinun Tech, Israel</td><td>r0080n370465</td><td>Nylon&amp;nbsp;screen aperture</td></tr><tr><td>Nylon micro-sieve with mesh size o 65 &amp;micro;m (size of 30 x 30 cm)</td><td>Sinun Tech, Israel</td><td>r0065n340715</td><td>Nylon&amp;nbsp;screen aperture</td></tr><tr><td>Nylon micro-sieve with mesh size o 50 &amp;micro;m (size of 30 x 30 cm)</td><td>Sinun Tech, Israel</td><td>r0080n370465</td><td>Nylon&amp;nbsp;screen aperture</td></tr><tr><td>Up to 10 g of frozen plant material (stored in -80 &lt;sup&gt;o&lt;/sup&gt;C or liquid nitrogen)</td><td></td><td></td><td></td></tr><tr><td>Suitable gloves for handling low temperatures</td><td></td><td></td><td></td></tr><tr><td>Safety goggles</td><td></td><td></td><td></td></tr><tr><td>1 mm screen door (mosquito) mesh (strip of 30 x 100 cm)</td><td></td><td></td><td></td></tr><tr><td>Large strainer (colander) with holes approximately 5 mm</td><td></td><td></td><td></td></tr><tr><td>1 L glass beaker</td><td></td><td></td><td></td></tr><tr><td>1 block of dry ice (0.5-1 kg)</td><td></td><td></td><td></td></tr><tr><td>Hammer and hard flat object</td><td></td><td></td><td></td></tr><tr><td>Two 5 L plastic containers</td><td></td><td></td><td></td></tr><tr><td>Rubber bands</td><td></td><td></td><td></td></tr><tr><td>Large flour sifter or sieve strainer- preferably one with a detachable plastic ring on the circumference</td><td></td><td></td><td></td></tr><tr><td>Several large and small round bottom stainless steel containers. One of them should be larger than the flour sifter&#039;s circumference (approximately 40 cm in diameter), to minimize the loss of the sifted mass outside the round bottome stainless steel container</td><td></td><td></td><td></td></tr><tr><td>Pre-chilled (via liquid nitrogen) stainless steel spoon, spatula, and scoopula</td><td></td><td></td><td></td></tr><tr><td>Clean plate</td><td></td><td></td><td></td></tr><tr><td>Several clothespins</td><td></td><td></td><td></td></tr><tr><td>Pre-chilled (via liquid nitrogen) labeled 1.5 mL tubes with holes poked on the lid with a sterile needle</td><td></td><td></td><td></td></tr><tr><td>Two containers of liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>1 cm wide painting brush</td><td></td><td></td><td></td></tr></tbody></table>

non-aqueous isolation and enrichment of glandular capitate stalked and sessile trichomes from cannabis sativa

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.

The main modification included in this protocol compared to conventional trichome isolation protocols is substituting the standard isolation medium with LN. Using LN as an isolation medium allows a relaxed workflow, because as long as the samples are submerged in LN, metabolic degradation is not likely to occur. Furthermore, as the protocol avoids the hazardous components (i.e., aurintricarboxylic acid and &#946;-mercaptoethanol) used in traditional trichome isolation medium, the work is not restricted to a chemical hood...

Watch this Scientific Journal Video about Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa at JoVE.com

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

A protocol is presented for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The protocol is based on a dry, non-buffer extraction of trichomes using only liquid nitrogen,&#160;dry ice, and nylon sieves and is suitable for RNA extraction and transcriptomic analysis.

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from ...

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JoVE Journal

Genetics

1.9K Views.  ARO-Volcani Center. Het belang van het protocol ligt in het gemak ervan in de korte tijd die nodig is voor de voltooiing ervan. Dit maakt een hoge doorvoer, een hoge concentratie en een hoge mate van isolatie van de gesteelde en sessiele trichomen mogelijk. Het belangrijkste voordeel van deze techniek is de rechtlijnigheid.Tijdens deze techniek moet men rekening houden met de trichoomdichtheid van de plantenbron en de isolatiegraad bij elke stap met een microscoop inspecteren terwijl het protocol voor de ...

Video: Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Immunology and Infection

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Bioengineering

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biology

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Comprehensive Approach for Microbial Isolation from Hidradenitis Suppurativa Tunnels

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's dermal layers, causing significant discomfort, foul-smelling discharge, disfigurement, contractures, and scarring, which severely diminish the quality of life. HS is associated with alterations in the skin microbiome, impacting immune regulation and the skin's defense against harmful bacteria. Despite its prevalence, the contribution of the HS microbiome to disease pathology and the limited response to treatment remains largely unknown. To date, multiple 16S rRNA sequencing studies on HS tissue have only achieved genus-level granularity, identifying an increase in Gram-negative anaerobes and a decrease in skin commensals. A deeper understanding of microbial dysbiosis in individuals with HS is essential for optimizing treatment strategies. This requires a two-pronged approach to assessing the HS microbiome, including the isolation of bacterial species, which are often underutilized in translational studies focused on skin disorders. Isolating individual microorganisms from HS tissue is crucial for elucidating the role of bacteria in HS pathogenesis. Here, we highlight reproducible methods to successfully isolate anaerobic pathogens from HS tunnel tissue, providing the initial and most critical step in understanding bacterial role in HS. This method paves the way for targeted research into microbial contributions to HS and for developing more effective, personalized treatment strategies that address the complex microbial burden of this chronic condition.

We showcase a method to successfully isolate fastidious and anaerobic organisms from cutaneous sinus tracts (tunnels) in tissues excised from patients with Hidradenitis Suppurativa.

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

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