Name: separation of avian preovulatory follicle granulosa and theca cell layers for downstream applications
Uploaded: 2024-10-25T14:40:00.000+00:00
Duration: 5 min 4 s
Description: 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 ...

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

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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.

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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

446 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

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

Biochemistry

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Developmental Biology

Gene Expression Analyses in Human Follicles

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff&#39;s laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 &#176;C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 &#176;C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 &#181;m) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/&#181;L. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

Here, we describe a protocol outlining how to isolate human ovarian follicles from frozen-thawed cortical tissue to perform gene expression analyses.

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization ...

Improved Procedure to Obtain Granulosa Cell-Free Stage I Zebrafish Oocytes

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model organism to investigate early developmental processes from oocyte to embryo. In zebrafish, oocytes are surrounded by a single layer of somatic granulosa cells. However, separating granulosa cells from oocytes poses a challenge, as achieving pure oocytes is crucial for precise analysis. Although various methods have been proposed to isolate zebrafish oocytes at different developmental stages, current techniques fall short in removing granulosa cells completely, limiting the accuracy of genome analysis focused solely on oocytes. In this study, we successfully developed a rapid and efficient process for isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. This technique facilitates biochemical and molecular analysis, particularly in exploring epigenetic and genome structure aspects specific to oocytes. Notably, the method is user-friendly, minimizes oocyte damage, and provides a practical solution for subsequent research and analysis.

Author Spotlight: Enhanced Method for Isolating Pure Zebrafish Stage I Oocytes

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific research.

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model ...

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 ...

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

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

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Rozdziały w tym wideo

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Gene Expression Analyses in Human Follicles

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

Absolute Quantum Yield Measurement of Powder Samples

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

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

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles