We are grateful to Milos Markis (AviServe) for assistance with animal husbandry, Nicole Guarino (University of Delaware) for assistance with manuscript preparation, Evelyn Weaver and Ramesh Ramachandran (The Pennsylvania State University) for assistance with procedure demonstration and manuscript preparation. Figure 2A and Figure 3 were created using BioRender.com using an institutional license sponsored by the University of Delaware Research Office. This work was supported by the UD CANR Comparative Pathology Laboratory. KME is supported by USDA NIFA grant 2023-67011-40333. This work was supported by grants from the University of Delaware Research Foundation (UDRF) and the Delaware INBRE program (supported by a grant from the National Institute of General Medical Sciences - NIGMS P20 GM103446 from the National Institutes of Health and the State of Delaware) to AD.

All animals used in this study were maintained and euthanized per the protocol approved by the University of Delaware&#39;s Institutional Animal Care and Use Committee (IACUC Protocol 110R).
1. Preparation
<ol>
	<li>Sterilization
	<ol>
		<li>Autoclave 5 oz glass bowls using gravity displacement autoclaving (121 &#176;C for 20 min) covered with aluminum foil with autoclave tape atop to maintain and indicate sterility. 
		NOTE: One bowl will be needed for each follicle the separation is being performed on.</li>
		<li>Autoclave dissection tools using gravity displacement autoclaving (121 &#176;C for 20 min), including scissors, curved forceps, and straight forceps in sterilization pouches.</li>
	</ol>
	</li>
	<li>Setup of separation area
	<ol>
		<li>Set up follicle separation area as seen in Figure 4. Fill holding containers with ice just prior to follicle separation and replenish as necessary throughout the follicle separation process.</li>
	</ol>
	</li>
	<li>Solutions
	<ol>
		<li>Dilute to 10x PBS concentration to 1x working stock and place in +4 &#176;C refrigerator the day prior to dissection.</li>
		<li>Dilute isopropanol to 70% working stock concentration in the spray bottle.</li>
	</ol>
	</li>
</ol>
2. Dissection
<ol>
	<li>Euthanize a sexually mature female bird.
	<ol>
		<li>Choose a sexually mature female bird (must be currently laying eggs) and euthanize16 by approved method (AVMA Guidelines) as per institutional care and use protocol, such as cervical dislocation.</li>
		<li>For the capture of the largest F1 follicle, euthanize the animal within the first 4 h of light onset for the day. Palpate the lower abdomen prior to euthanasia to confirm a hard-shelled egg in the uterus; this ensures that oviposition and, thus, the next ovulation has not yet occurred.</li>
	</ol>
	</li>
	<li>Access the ovary.
	<ol>
		<li>Spray the external carcass and feathers with 70% isopropanol. This dampens the feathers and cleans the incision point.</li>
		<li>Using dissection scissors and a gloved hand, pinch and cut a single horizontal (transverse) midline incision approximately 7.5 cm (3 inches) long. Pinch the skin, muscle, and fat layers up prior to incision to avoid puncture of organs.</li>
		<li>Widen the incision laterally on the bird&#39;s right side and probe with gloved fingers to push visceral organs out of the way of the scissors. Continue until 2-3 of the animal&#39;s ribs have been cut on the right side.</li>
		<li>Widen the incision toward the left side of the bird as done on the right.&#160;Proceed with added caution as the preovulatory follicles of the ovary are located here, protrude from the ovary surface, and can easily rupture. Use gentle pressure with fingers to keep preovulatory follicles away from the scissors as 2-3 ribs are cut on the bird&#39;s left side.</li>
		<li>Place one hand on the lower abdomen of the bird and the other firmly on the underside of the breastbone, and manually retract the breast of the bird upward to allow further access to the coelomic cavity of the bird.</li>
		<li>Holding each of the bird&#39;s thighs close to the hock joint, manually push them backward to dislocate both hip joints simultaneously. There is now complete access to the ovary both visually and spatially.</li>
	</ol>
	</li>
	<li>Remove preovulatory follicles.
	<ol>
		<li>Perform all steps by routine visual sight. If an individual has trouble visualizing at any stage, use a dissecting microscope.</li>
		<li>Using only gloved fingers, gently cup each visible yellow preovulatory follicle and extract it from the ovary. Only pinch at the stalk during the extraction to assist in severing the stalk. Do not pinch the follicle itself, or it may rupture.</li>
		<li>Place each follicle into the sterile container with ice-cold PBS.</li>
		<li>Leave preovulatory follicles on ice for at least 5 min to allow for congealing of the yolk. The warm yolk will adhere to the surrounding follicular cell layers, making the subsequent separation techniques difficult to perform.</li>
	</ol>
	</li>
</ol>
3. In-lab separation
<ol>
	<li>Take an isolated follicle from the sterile container and place the container with the remaining follicles back on ice. Study the follicle prior to manipulation, taking note of the location of three features: the stalk, the stigma, and the germinal disc (Figure 2).</li>
	<li>Begin by removing the serosa from the follicular surface. Roll the follicle gently over a dry paper towel to remove the serosal layer, gently pulling it off if necessary.</li>
	<li>Hold the follicle by the stalk over the bowl filled with PBS. Use gravity to visualize where the stalk ends at the surface of the follicle and, using scissors, snip only the stalk without puncturing the follicle surface. Continue with the removal of the serosa until it is no longer visible.</li>
	<li>Using the lightbox to illuminate and the germinal disc as a reference, relocate the stigma on the opposite side and gently hold the follicle with the stigma side visible. Hold the follicle directly over the bowl of ice-cold PBS.</li>
	<li>Pick up the scalpel, slice gently along the length of the stigma, and immediately drop the follicle into the PBS as the yolk begins to fall out. Do not&#160;hold the follicle tightly, as this will push the yolk out instead of allowing it to simply fall out through gravity.</li>
	<li>Using a pair of straight untoothed tissue forceps, gently peel back the pink layer of the follicular wall from the yolk on one side of the slice. Complete this extraction using little pinch-and-pull motions that lift the follicle wall away from the yolk.</li>
	<li>Layer separation works best when approximately 5 mm of follicular wall tissue has been retracted away from the yolk at a time. When a 5 mm section of the follicular wall is retracted successfully from the yolk, use angled forceps in tandem with the straight forceps to probe along the follicle shell.</li>
	<li>In some instances, the granulosa layer may be more closely associated with the yolk than the theca layer. In this situation, simply probe very gently across the surface of the yolk using either pair of forceps to try to pull the tissue-paper-like layer away from the yolk. Once the granulosa layer has been located, pinch it securely to the theca layer and proceed with pulling both layers away from the yolk as previously described.</li>
	<li>Use the curved forceps to lightly brush the yolk away from the pinched layers. Sweep away or remove excess yolk from the bowl. Do not scrape the granulosa layer; this will cause tearing. Vigorous motions will cloud the PBS solution and reduce visualization. Proceed with careful and deliberate motions.</li>
	<li>After each 5-10 mm section of follicular wall is retracted, pause to take the theca and granulosa layers in each set of forceps and ensure that the two layers are being peeled away from each other gradually, just as they are both being peeled away from the yolk. Continue this process around the outside of the follicle. If manipulation becomes difficult due to the flap of the granulosa-theca layer, turn the follicle to begin the process from the other side of the slice.</li>
	<li>During the process of separation, the germinal disc (female gamete and germinal vesicle) will be encountered. This is an area that is extremely adherent to both the granulosa layer and the yolk. Approach the germinal disc carefully, using the straight forceps to firmly hold the granulosa layer to the theca layer, as the curved forceps are used in light brushing motions to push the germinal disc away from the granulosa cells onto the yolk surface.
	<ol>
		<li>If the germinal disc is desired as a research specimen, brush off the yolk at this point with angled forceps and store as desired.</li>
	</ol>
	</li>
	<li>Continue separating the yolk from the follicular wall with the curved forceps until the entire sphere of the follicle has had its yolk removed.</li>
	<li>Once the remainder of the yolk has been brushed from the layers, separate the last portion of the granulosa layer from the theca layer. The fully separated granulosa layer may not be entirely devoid of yolk matter, but ensure it is not coated with it. Place the granulosa layer and thecal layer into separate containers with 4 &#176;C temperature PBS.
	<ol>
		<li>Once the granulosa layer is in a separate container from the theca layer, gently agitate the theca layer back and forth in the PBS with forceps to ensure any remaining remnants of the granulosa layer detach. 
		NOTE: Certain processing methods post-separation may account for the removal of lipid debris by digestion or centrifugation, depending on the desired study.</li>
		<li>When first practicing this technique, prepare and analyze representative separated specimens histologically to confirm accurate layer separation and collection.
		<ol>
			<li>To do this, fix portions of separated tissues in neutral buffered formalin using mesh tissue cassettes or sponges to prevent specimen loss, then paraffin-embed, section, and stain the section with Hematoxylin and Eosin (H&#38;E).</li>
			<li>Due to its delicate nature, carefully handle and place the granulosa layer onto biopsy sponges in a histological cassette to ensure the cellular layer stays intact.</li>
			<li>Put the theca layer directly within a histological cassette. Doing so will also allow visualization of the level of specimen contamination by other follicular tissue (i.e., granulosa cell layer specimen with adherence of yolk protein or thecal cells). 
			NOTE: It may not always be possible to obtain only a single layer of interest due to the close association of follicular tissues. However, knowing the level of possible contamination can inform the interpretation of subsequent multi-omics approaches.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Separation of Hen Ovarian Follicle Layers for Reproductive Research Applications

<ol>
	<li>Finter, N. B., Liu, O. C., Henle, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+host-virus+interactions+in+the+chick+embryo-influenza+virus+system.+X.+An+experimental+analysis+of+the+von+Magnus+phenomenon.">Studies on host-virus interactions in the chick embryo-influenza virus system. X. An experimental analysis of the von Magnus phenomenon.</a> J Exp Med. 101 (5), 461-478 (1955).</li><li>Fredrickson, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+tumors+of+the+hen.">Ovarian tumors of the hen.</a> Environ Health Perspect. 73, 35-51 (1987).</li><li>Etches, R. J., Petitte, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reptilian+and+avian+follicular+hierarchies:+models+for+the+study+of+ovarian+development.">Reptilian and avian follicular hierarchies: models for the study of ovarian development.</a> J Exp Zool Suppl. 4, 112-122 (1990).</li><li>McCarrey, J. R., Abbott, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+genetic+sex+determination,+gonadal+sex+differentiation,+and+germ-cell+development+in+animals.">Mechanisms of genetic sex determination, gonadal sex differentiation, and germ-cell development in animals.</a> Adv Genet. 20, 217-290 (1979).</li><li>Tilly, J. L., Kowalski, K. I., Johnson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage+of+ovarian+follicular+development+associated+with+the+initiation+of+steroidogenic+competence+in+avian+granulosa+cells.">Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.</a> Biol Reprod. 44 (2), 305-314 (1991).</li><li>Gilbert, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+the+ovarian+follicle+of+the+domestic+hen.">Innervation of the ovarian follicle of the domestic hen.</a> Q J Exp Physiol Cogn Med Sci. 50 (4), 437-445 (1965).</li><li>Johnson, P. A., Stoklosowa, S., Bahr, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+granulosa+and+theca+layers+in+the+control+of+progesterone+secretion+in+the+domestic+hen.">Interaction of granulosa and theca layers in the control of progesterone secretion in the domestic hen.</a> Biol Reprod. 37 (5), 1149-1155 (1987).</li><li>Apperson, K. D., Bird, K. E., Cherian, G., Lohr, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+of+the+ovary+of+the+laying+hen+(Gallus+domesticus).">Histology of the ovary of the laying hen (Gallus domesticus).</a> Vet Sci. 4 (4), 66 (2017).</li><li>Johnson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicle+selection+and+granulosa+cell+differentiation.">Ovarian follicle selection and granulosa cell differentiation.</a> Poult Sci. 94 (4), 781-785 (2015).</li><li>Kim, D., Johnson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+granulosa+layer+from+hen+prehierarchal+follicles+associated+with+follicle-stimulating+hormone+receptor+signaling.">Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle-stimulating hormone receptor signaling.</a> Mol Reprod Dev. 85 (8-9), 729-737 (2018).</li><li>Lu, 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=Effects+of+exogenous+energy+on+synthesis+of+steroid+hormones+and+expression+characteristics+of+the+CREB/StAR+signaling+pathway+in+theca+cells+of+laying+hen.">Effects of exogenous energy on synthesis of steroid hormones and expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen.</a> Poult Sci. 103 (3), 103414 (2024).</li><li>Huang, Y., 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=Comparative+analysis+among+different+species+reveals+that+the+androgen+receptor+regulates+chicken+follicle+selection+through+species-specific+genes+related+to+follicle+development.">Comparative analysis among different species reveals that the androgen receptor regulates chicken follicle selection through species-specific genes related to follicle development.</a> Front Genet. 12, 752976 (2021).</li><li>Tilly, J. L., Johnson, A. L. <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+androstenedione+production+by+adenosine+3',5'-monophosphate+and+phorbol+myristate+acetate+in+ovarian+thecal+cells+of+the+domestic+hen.">Regulation of androstenedione production by adenosine 3',5'-monophosphate and phorbol myristate acetate in ovarian thecal cells of the domestic hen.</a> Endocrinology. 125 (3), 1691-1699 (1989).</li><li>Young, J. M., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theca:+the+forgotten+cell+of+the+ovarian+follicle.">Theca: the forgotten cell of the ovarian follicle.</a> Reproduction. 140 (4), 489-504 (2010).</li><li>Gilbert, A. B., Evans, A. J., Perry, M. M., Davidson, 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=A+method+for+separating+the+granulosa+cells,+the+basal+lamina+and+the+theca+of+the+preovulatory+ovarian+follicle+of+the+domestic+fowl+(Gallus+domesticus).">A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus).</a> J Reprod Fertil. 50 (1), 179-181 (1977).</li><li>AVMA Panel on Euthanasia. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA guidelines for the euthanasia of animals: 2020 edition. , (2020).</li><li>Mori, M., Kohmoto, K., Shoda, Y. <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+granulosa+and+theca+cells+on+in+vitro+progesterone+production+in+preovulatory+follicles+of+the+Japanese+quail.">Role of granulosa and theca cells on in vitro progesterone production in preovulatory follicles of the Japanese quail.</a> Japan Poult Sci. 21 (4), 206-214 (1984).</li><li>Porter, T. E., Hargis, B. M., Silsby, J. L., El Halawani, M. E. <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+dissimilar+steroid+productions+by+granulosa,+theca+interna+and+theca+externa+cells+during+follicular+maturation+in+the+turkey+(Meleagris+gallopavo).">Characterization of dissimilar steroid productions by granulosa, theca interna and theca externa cells during follicular maturation in the turkey (Meleagris gallopavo).</a> Gen Comp Endocrinol. 84 (1), 1-8 (1991).</li><li>Bahr, J. M., Wang, S. C., Huang, M. Y., Calvo, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+concentrations+in+isolated+theca+and+granulosa+layers+of+preovulatory+follicles+during+the+ovulatory+cycle+of+the+domestic+hen.">Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen.</a> Biol Reprod. 29 (2), 326-334 (1983).</li><li>Micke, P., 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=Biobanking+of+fresh+frozen+tissue:+RNA+is+stable+in+nonfixed+surgical+specimens.">Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens.</a> Lab Invest. 86 (2), 202-211 (2006).</li><li>Madisen, L., Hoar, D. I., Holroyd, C. D., Crisp, M., Hodes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+banking:+the+effects+of+storage+of+blood+and+isolated+DNA+on+the+integrity+of+DNA.">DNA banking: the effects of storage of blood and isolated DNA on the integrity of DNA.</a> Am J Med Genet. 27 (2), 379-390 (1987).</li><li>Yang, Y. Z., 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=Histological+characteristics+of+follicles+and+reproductive+hormone+secretion+during+ovarian+follicle+development+in+laying+geese.">Histological characteristics of follicles and reproductive hormone secretion during ovarian follicle development in laying geese.</a> Poult Sci. 98 (11), 6063-6070 (2019).</li><li>Wang, Y., 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=Quantitative+proteomic+analyses+during+formation+of+chicken+egg+yolk.">Quantitative proteomic analyses during formation of chicken egg yolk.</a> Food Chem. 374, 131828 (2022).</li><li>Vanmontfort, D., Rombauts, L., Decuypere, E., Verhoeven, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Source+of+immunoreactive+inhibin+in+the+chicken+ovary.">Source of immunoreactive inhibin in the chicken ovary.</a> Biol Reprod. 47 (6), 977-983 (1992).</li></ol>

The authors have no competing interests to report.

This study outlines the procedure for the distinct separation of both granulosa and thecal cell layers from preovulatory follicles in poultry. Unlike the existing method established by Gilbert et al. in 1977, the adaptations made in this method allow for a more controlled environment when manually separating the granulosa cell layer from the theca layer from all preovulatory follicles15. Furthermore, it enables researchers to visualize the granulosa cell layer throughout the whole procedure, enhan...

Egg-producing chickens are a key component of the world food supply chain and are an evolving animal model for the study of fertility and ovarian cancer. The chicken has a long history of laboratory use, from in ovo studies of influenza vaccines1 to the study of cancerous tumor growth2, and is a key animal for insight into gonadal development3,4,5. Understanding the role of a hen&#39;s reproductive system, in particular, the cyclical nature of ovarian follicular development and the types of cells involved, is an area of great interest and potential application. Multi-omics approaches to interrogate the hen ovary have applications for increasing agricultural yield through productivity analysis and elucidating factors toward human health by way of the hen as a model for reproductive disease.
The avian ovary has very distinct developmental follicular stages composed of (1) small primordial and primary follicles (&#62;1 mm), (2) variably sized pre-recruitment follicles (white to pale yellow, 2-8 mm), and (3) large, yolk-filled preovulatory follicles (yellow) (Figure 1)5,6,7. A sexually mature avian ovary is composed of an outer cortex that houses the ovarian follicles and the inner medulla composed of smooth muscle, nerves, and vasculature8. The entire cycle of follicular development takes place in the cortex. It is not until approximately five months of age, however, that only the left ovary demonstrates a full developmental hierarchy of preovulatory (i.e., yellow) follicles, and the chicken will undergo her first ovulation and oviposition (egg lay). The preovulatory follicles establish a grossly visible hierarchy of approximately 5 to 6 follicles that range from the largest and next to ovulate - F1 follicle - to the lesser developed, smallest follicle- F5 or F6. Preovulatory follicles are easily distinguishable from the smaller pre-recruitment follicles due to their larger size, vast innervation, and deep-yellow yolk.
All follicles contain specialized cell types that support varied roles in signaling, growth, and development of the follicle as it progresses through different stages prior to ovulation - these are the granulosa cells, theca cells, and oocytes (female gametes) (Figure 2A). In particular, the granulosa cell layers create the inner wall of the follicle, and the theca cells form the other wall surrounding the oocyte. As a follicle progresses through the preovulatory hierarchy, the theca and granulosa cell layers begin to thin out, particularly along a point directly across from the stalk of the preovulatory follicle. The stalk is the follicle&#39;s point of attachment to the ovary (Figure 2B). This line of the granulosa and theca layers opposite the stalk is called the stigma (Figure 2C). It entirely lacks vasculature and will be the point of follicular rupture during ovulation of an F1 follicle. Also visible is the germinal disc, or the fluffy white layer of support cells that surround the oocyte (Figure 2B).
Both granulosa and theca cells are key components of follicle development as they surround and support the oocyte through complex hormonal signaling. The granulosa cell layer supports steroidogenic pathways via cyclic AMP (cAMP) signaling to bring about progesterone production9,10,11. Theca cells, on the other hand, temporally produce estradiol, which is largely different than in mammals whose theca cells produce androgen and progesterone12,13,14. Taken together with external signaling, the granulosa and theca cells are crucial drivers in follicle maturation and oviposition. The technique of separating the granulosa and theca layers was originally reported by Gilbert et al. in 197715. Achieving a comprehensive separation of the granulosa and theca layers and ensuring no granulosa material remains behind can present difficulties with the Gilbert method. Furthermore, the absence of a clear visual guide for completing the separation can render the task challenging to conduct. This study aims to describe the extraction and separation of granulosa, theca, and oocyte layers from the preovulatory follicles, offering adaptations and delivering a clear visual representation through the use of both images and videos (Figure 3). Visualizing this technique will allow scientists studying avian ovarian follicles to reproducibly capture granulosa and theca layers for both agricultural and biomedical multi-omics approaches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3.5 in. Small Glass Bowls, 5 oz</td>
			<td>Amazon</td>
			<td>B0BXP5PJTN</td>
			<td>Autoclavable glass bowls of at least 3.5 in. diameter</td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>Costco</td>
			<td>720</td>
			<td>Cover autoclave bowls</td>
		</tr>
		<tr>
			<td>Amazon Pet Training Pads, Regular, 100-Count</td>
			<td>Amazon</td>
			<td>B0B58WTPFS</td>
			<td>For use as necrospy pads, any absorbant pad will work</td>
		</tr>
		<tr>
			<td>Autoclave Tape</td>
			<td>Fisherbrand</td>
			<td>15-901-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Cole-Parmer LED Fiber Optic Illuminators</td>
			<td>Cole-Parmer</td>
			<td>EW-41723-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Very Fine Precision Tip Forceps</td>
			<td>Fisherbrand</td>
			<td>16-100-123</td>
			<td>Non-Serrated</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Leica&#160;</td>
			<td>S6E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Precision Scissors</td>
			<td>Fisherbrand</td>
			<td>12-000-155</td>
			<td>Non-Serrated</td>
		</tr>
		<tr>
			<td>Food Container Boxes with Lid Set of 17 Clear/Green Microwave Freezer Dishwasher Safe</td>
			<td>Amazon</td>
			<td>B09QGZCRDB</td>
			<td>Use any container that can hold ice AND an 3.5 in. small glass bowl</td>
		</tr>
		<tr>
			<td>High Precision 45 Degree Curved Tapered Very Fine Point Tweezers/Forceps</td>
			<td>Fisherbrand</td>
			<td>12-000-125</td>
			<td>Non-Serrated</td>
		</tr>
		<tr>
			<td>High Precision Straight Very Fine Point Tweezers/Forceps</td>
			<td>Fisherbrand</td>
			<td>16-100-120</td>
			<td>Non-Serrated</td>
		</tr>
		<tr>
			<td>Isopropanol&#160;</td>
			<td>Fisherbrand</td>
			<td>A426P-4</td>
			<td></td>
		</tr>
		<tr>
			<td>McKesson Specimen Container, Sterile, Screw Cap, Leak-Resistant, 120 mL</td>
			<td>McKesson</td>
			<td>16-9526</td>
			<td>4 oz. / 120 cc, graduated</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS, 10x), pH 7.6</td>
			<td>ThermoFisher</td>
			<td>J62692.K7</td>
			<td>Will need to be made into 1x PBS</td>
		</tr>
		<tr>
			<td>Spray Bottle</td>
			<td>Cole-Parmer</td>
			<td>EW-06091-01</td>
			<td>For 70% Isopropanol</td>
		</tr>
		<tr>
			<td>Sterile Surgical Blades #22</td>
			<td>Cincinnati Surgical</td>
			<td>122</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization Pouches 10&#34; x 16&#34;</td>
			<td>Amazon</td>
			<td>B07MFB455C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered Ultrafine Tip Forceps Fisherbrand&#160;</td>
			<td>Fisherbrand</td>
			<td>16-100-121</td>
			<td>Non-Serrated</td>
		</tr>
		<tr>
			<td>Foam Biopsy Pads, Rectangular</td>
			<td>Fisherbrand</td>
			<td>22-038-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin Solution, 10% (Histological)</td>
			<td>Fisher Chemical</td>
			<td>SF98-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processing/embedding cassettes with lid</td>
			<td>Simport M490-2</td>
			<td>Z672122</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of avian preovulatory follicle granulosa and theca cell layers for downstream applications

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. 
This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.

This protocol yields the separation of granulosa and thecal cell layers from the ovarian follicle (Figure 5 and Figure 6). These layers can then be prepared for histological examination to confirm successful separation. These histological images, reviewed and captured by a board-certified veterinary pathologist (EMB), clearly demonstrate the effective separation of the granulosa and theca layers for both the F1 and F5 preovulatory follicles (<strong class="xfig"...

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable ...

separation-avian-preovulatory-follicle-granulosa-theca-cell-layers

Research

JoVE Journal

Biology

358 Views.  University of Delaware. Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights. 

Video: Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...