This work was supported by a grant from the National Science Council Taiwan (NSC102-2320-B-037-008-MY3) and a National Cheng-Kung University Hospital Research Grant (NCKUH-10102045).

All the procedures including animal subjects have been approved by Institutional Animal Care and Use Committee of Kaohsiung Medical University.
1. Experimental Procedure
<ol>
	<li>Sacrifice the adult (8&#160;to 12 week-old) female/male SD rats by CO2 euthanasia.</li>
	<li>Apply 70% ethanol and let it be absorbed into the skin, and then wipe the skin with a tissue paper.</li>
	<li>Put the rat on a clean plastic plate in the supine position.</li>
	<li>Lift the skin with &#8220;external&#8221; curved forceps at the midline and perform a blunt dissection by creating a &#8220;Y&#8221; shape (from the level of the thigh to the subcostal angle).
	<ol>
		<li>Rinse scissors with 70% ethanol and, then, cut the muscle, also creating a &#8220;Y&#8221; shape.</li>
	</ol>
	</li>
	<li>Find the left adrenal gland behind the stomach. 
	NOTE: The right adrenal can be found deeply behind the liver.
	<ol>
		<li>Use one pair of forceps to lift the stomach or liver and isolate the adrenals with another curved forceps.</li>
	</ol>
	</li>
	<li>After putting the adrenal glands into a sterile dish, carefully remove the adrenal capsule with fine forceps.</li>
	<li>With a delicate pair of scissors, cut the adrenals into small pieces with a little bit of serum-free Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)/F-12 medium.</li>
	<li>Prepare three different pore-sized Pasteur pipets and use the first Pasteur pipet to aspirate all the adrenal pieces into a 50 mL conical tube containing medium with collagenase II (~0.5&#8211;0.8 mg/mL).
	<ol>
		<li>Gently triturate the tissue pieces about 10x. Use the rest of the pipets and repeat the above steps until all the pieces become smaller. 
		NOTE: The different pore sizes of the Pasteur pipets can be created by heating them with fire. The first pipet is the original size (about 1 mm), the pore size of the second one is about 0.8 mm, and the third Pasteur pipet is about 0.5 mm. During digestion, the pieces of adrenal will become smaller. Thus, via the mechanical procedure, the adrenals can be digested more completed. Incubate the tube in a water bath (at 37 &#176;C) and shake it every 5 min, 4x in total.</li>
	</ol>
	</li>
	<li>After the 20 min incubation, add fresh and cool medium (sixfold volume; for example, 5 mL of tissue + 25 mL of cool DMEM/F-12 medium) containing 2.5% fetal bovine serum (FBS) and 5% horse serum to stop the enzyme effect.</li>
	<li>Collect the cell pellets by centrifugation at 800 x g&#160;for 10 min.</li>
	<li>Add an appropriate volume of medium to suspend the cells. 
	NOTE: Each gland can make about ~3 x 105 to ~4 x 105 cells.</li>
	<li>Plate the cells in desired plates or dishes with growth medium (1:1 [v/v] mixture of DMEM and Ham&#8217;s F-12 medium, supplemented with 25 mM HEPES and 1% penicillin and streptomycin) overnight (day 0) at 37 &#176;C, in a humidified environment of 95% air and 5% CO2. 
	NOTE: Usually, we plate cells in 24-well plates (four glands/plate) for the corticosterone assay and in 35 mm dishes (one gland/approximately one to two dishes) for western blot analysis.</li>
	<li>Carefully wash the cells 2x with serum-free warm DMEM/F-12 medium (day 1). Replace the new growth medium.</li>
	<li>Maintain the cells at 37 &#176;C in a humidified environment of 95% air and 5% CO2 until day 3.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenal+glands+of+mouse+and+rat+do+not+synthesize+androgens.">Adrenal glands of mouse and rat do not synthesize androgens.</a> Life Sciences. 50 (12), 857-861 (1992).</li><li>Wang, T., Rainey, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adrenocortical+carcinoma+cell+lines.">Human adrenocortical carcinoma cell lines.</a> Molecular and Cellular Endocrinology. 351 (1), 58-65 (2012).</li><li>Payne, A. H., Hales, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+steroidogenic+enzymes+in+the+pathway+from+cholesterol+to+active+steroid+hormones.">Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones.</a> Endocrine Review. 25 (6), 947-970 (2004).</li><li>Ruggiero, C., Lalli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+ACTH+Signaling+on+Transcriptional+Regulation+of+Steroidogenic+Genes.">Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes.</a> Frontiers in Endocrinology. 7, 24 (2016).</li><li>Chen, Y. C., Liang, Y. L., Huang, Y. L., Huang, B. 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The authors have nothing to disclose.

Adrenal glands play a key role in environmental adaptation3. The hormone by which the adrenal cortex secretes can regulate and adjust physiological functions and homeostasis3. The in vivo&#160;model reflects the real physiological effects in the body. However, it is still influenced by many complex factors, causing unstable effects in experiments. In contrast, the in vitro cell model has advantages which makes it incomparable to the animal model. First, the environmental co...

The endocrine system is responsible for regulating physiological activities and homeostasis1. Adrenal glands located at the cranial pole of the kidneys are one of the major endocrine organs which secrete mineralocorticoids, glucocorticoids, and androgen2,3. There are two distinct parts of the adrenal gland: the cortex and the medulla. The adrenal cortex consists of three layers: the outer glomerulosa, the intermediate fasciculata, and the inner reticularis3. The zona glomerulosa is the original secreting site of aldosterone, a mineralocorticoid, which aids in reabsorbing sodium ion and water in the kidneys3. The zona fasciculata primarily produces a basal level of glucocorticoids in normal physiological conditions3. Corticosteroids in rodents and cortisol in humans act to assist the body in coping with stress by regulating blood glucose3. To some extent, they can inhibit inflammatory responses and regulate the immune system4,5. In contrast to other mammals, mice and rats do not have a functional zona reticularis due to the lack of a 17&#945;-hydroxylase expression in the adrenal gland6,7. Thus, adrenals from mice and rats are devoid of the secretion of adrenal C-19 steroids (cortisol and adrenal androgens).
Primary cultured adrenocortical cells have been shown to be useful for investigating mechanisms controlling the adrenal physiology8. Like other steroidogenic tissues, each adrenal zone synthesizes its steroids from free cholesterol9. Upon adrenocorticotropic hormone (ACTH) stimulation, the free cholesterol is released from breakdown lipid droplets and, thus, enhances steroid production in the fasciculata and reticularis10.
Many groups have attempted to establish stable cell lines from adrenocortical carcinomas. However, several concerns have limited the use of adrenocortical cell lines as in vitro models8. The steroid responses of cell lines may differ between passages, the lot number of serum, and the quality of the serum, etc. Moreover, commercial adrenal cell lines (Y1 and SW13) do not secrete corticosterone, making it more difficult to study adrenal steroidogenesis8. Using bovine and equine adrenals as ex vivo models may be a good choice, even though tissues from these markets may obscure some unknown risks. Compared to them, managing rat adrenal glands is easier and the source is relatively more stable and pathogen-free. Taken together, the present method described here could be used to investigate the related mechanism of corticosterone synthesis in rat adrenal fasciculata cells.

<table><tbody><tr><td>Female/male, SD/Wistar rats (8-12 weeks)</td><td>BioLASCO</td><td></td><td>male or femal rats are suitable for experiments. Female&#039;s adrenal glands are larger than male rats, however, female rats own the oestrous cycles that may influence the experiments</td></tr><tr><td>Collagenase, type II</td><td>Sigma</td><td>C-6885</td><td>isolation of adrenal cells</td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>12800-017</td><td>powder media for adrenal cells</td></tr><tr><td>Ham&#039;s F12</td><td>ThermoFisher</td><td>21700-075</td><td>powder media for adrenal cells</td></tr><tr><td>HEPES</td><td>JT.baker</td><td>4018</td><td>buffer (powder)</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>JT.baker</td><td>3506-1</td><td></td></tr><tr><td>Horse serum</td><td>Hyclone</td><td>16050014</td><td></td></tr><tr><td>Fetal bovine serum</td><td>SAFC</td><td>12103C</td><td></td></tr><tr><td>Penicillin (10,000 U)-streptomycin (10,000 ug/mL)</td><td>Corning</td><td>30-002-Cl</td><td>antibiotics</td></tr><tr><td>Curved forceps</td><td>BRAUN</td><td>BD343R</td><td></td></tr><tr><td>Forceps</td><td>BRAUN</td><td>BD331R</td><td></td></tr><tr><td>Scissor</td><td>BRAUN</td><td>BC224R</td><td>cut rat skin and muscle</td></tr><tr><td>Delicate scissor</td><td>BRAUN</td><td>BC101R</td><td>cut adrenal glands</td></tr><tr><td>Adipose-differentiation related protein</td><td>Abcam</td><td>ab108323</td><td>antibody for lipid droplet associated protein</td></tr><tr><td>Corticosterone ELISA kit</td><td>cayman</td><td>500655</td><td>assay for corticosterone synthesis</td></tr><tr><td>Inverted light microscopy</td><td>Leica</td><td></td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>JT.baker</td><td>X198-07</td><td>for immunofluorescence staining</td></tr><tr><td>Goat anti-rabbit IgG Alexa 488 Fluor secondary antibody</td><td>ThermoFisher</td><td>A-11034</td><td>for immunofluorescence staining</td></tr><tr><td>Anti-ADFP</td><td>Abcam</td><td>108323</td><td>for immunofluorescence staining</td></tr><tr><td>ProLong Gold antifade mountant</td><td>ThermoFisher</td><td>P36935</td><td>for immunofluorescence staining</td></tr></tbody></table>

primary culture of rat adrenocortical cells and assays of steroidogenic functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

Using the procedure described here, primary cultured adrenal cells could be distinguished under a phase-contrast microscope (Figure 1A). To further confirm that the vesicles within the cytoplasm are lipid droplets, the immunofluorescence staining of adipose differentiation-related protein (ADRP) could be conducted on day-3 cultures (Figure 1B). Cells were grown on the glass coverslips. The cells were washed three times with phosp...

Watch this Scientific Journal Video about Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions at JoVE.com

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

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Biology

8.0K Views.  Kaohsiung Medical University. Het gepresenteerde protocol is zeer eenvoudig en het zal onderzoekers helpen bij te bereiden primaire bijnierculturen. Het biedt een gemakkelijke procedure voor het starten van bijniersteroïde. Deze techniek kan worden toegepast bij het screenen van de stress-gerelateerde ziekten, alle mogelijkheden van de agent-geremde ontstekingsreacties.Het aantonen van de procedure zal Wei-Chang Lee zijn, een Masters student uit mijn laboratorium. Begin met het steriliseren van euthanasized SD rat...

Video: Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

Mouse Adrenal Chromaffin Cell Isolation

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

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

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

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Developmental Biology

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Immunology and Infection

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

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