Name: Video: Kolekcja White and Brown Adipose Depot od Mouse Pup: procedura chirurgiczna mająca na celu zebranie białej tkanki tłuszczowej i brązowej tkanki tłuszczowej od szczeniaka myszy - Experiment
Uploaded: 2025-07-08T21:26:24.000+00:00
Duration: 3 min 28 s
Description: 4.6K Views. Źródło: Galmozzi, A. et al. Izolacja i różnicowanie pierwotnych białych i brązowych preadipocytów od nowonarodzonych myszy. J. Vis. Termin ważności (2021) W tym filmie pokazujemy procedurę chirurgiczną polegającą na izolowaniu podskórnej białej tkanki tłuszczowej i brązowej tkanki tłuszczowej między łopatkami od nowonarodzonej myszy.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Collection and digestion of adipose depots (day 1)
<ol>
	<li>Prepare two 1.5 mL tubes for each pup: one for brown adipose tissue (BAT) and one for white adipose tissue (WAT). Add 250 &#181;L of phosphate-buffered saline (PBS) + 200 &#181;L of 2x isolation buffer (123 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin-streptomycin, and 4% fatty acid-free bovine serum albumin) to each tube. Keep solutions sterile and on ice.</li>
	<li>Place the pups into small chambers (e.g., one well of a 6-well plate), and set them on ice until they become hypothermic. Make sure that there is no direct contact between the pups and the ice. Prick a paw with a tip to assure loss of consciousness and euthanize the pups by decapitation using sharp scissors. 
	NOTE: if working with different genotypes, prepare an additional tube to collect a tail biopsy (3 mm cut) for genotyping. If genotyping is performed immediately, keep the euthanized pups on ice until the dissection to collect adipose depots.</li>
	<li>Calculate and weigh out the amount of collagenase needed to digest all the depots. Add 50 &#181;L of 15 mg/mL collagenase type I in 2x isolation buffer to each tube. Do not resuspend the collagenase in isolation buffer until all the tissues have been collected.</li>
	<li>To collect subcutaneous WAT, cut the skin around the pup's abdomen (avoid peritoneal rupture), and gently pull the skin down below the legs. Without taking the skin, carefully collect the fat depot, which will appear as a clear (P1 or younger) or white (P2 and older), thin, elongated tissue attached on the inside or on top of the quadriceps (Figure 1A, B). Rinse the fat depot in PBS and place it in one of the tubes containing 250 &#181;L of PBS + 200 &#181;L of 2x isolation buffer. Keep on ice. 
	NOTE: P0 and P1 mice give the best yield. In P0-1 pups, the subcutaneous WAT depot is nearly transparent. In P2 mice and older, the depot is easier to identify because it is already turning white, indicating the presence of fully mature, lipid-loaded, white adipocytes.</li>
	<li>To collect interscapular BAT, pull the skin from the shoulder blades over the head. Lift the BAT - the deep red tissue between the shoulder blades - and carefully make incisions all around it to detach it from the body (Figure 1A, B). Check for consistency and color; rinse with PBS; place it in the other tube containing 250 &#181;L of PBS + 200 &#181;L of 2x isolation buffer; and keep on ice. 
	NOTE: When harvesting BAT from P2 mice and older, carefully remove the white adipose tissue surrounding the BAT. It consists of a thin, soft, white sheet of adipocytes located between the skin and the BAT, which is deeper between the shoulder blades.</li>
</ol>

<table><tbody><tr><td>6-well plates</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Fatty Acid-Free BSA</td><td>Sigma-Aldrich</td><td>A8806</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Surgical forceps</td><td>ROBOZ Surgical Instrument Co</td><td>RS-5158</td><td></td></tr><tr><td>Surgical Scissors</td><td>ROBOZ Surgical Instrument Co</td><td>RS-5880</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>&amp;nbsp;Gibco&amp;nbsp;</td><td>14190144</td><td></td></tr></tbody></table>

white and brown adipose depot collection from mouse pup: a surgical procedure to harvest the white adipose tissue and brown adipose tissue from mouse pup

Source: Galmozzi, A. et al. <a href='https://www.jove.com/v/62005'>Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the surgical procedure to isolate the subcutaneous white adipose tissue and interscapular brown adipose tissue from a newborn mouse.

<img alt="WAT and BAT processing diagram, mouse samples, fat tissue analysis, microscopic cell results." src="/files/ftp_upload/20802/20802_Figure1.jpg" data-altupdatedat="1753371239000" data-alt="Figure 1"> 
Figure 1: Collection and processing of fat pads. (A) Schematic representation of primary white (top) and brown (bottom) adipocyte isolation. (B) Subcutaneous white (top) and brown (bottom) adipose depots. In P0 mice, subcutaneous WAT is almost invisible, but becomes distinguishable on ~day 2 after birth. In contrast, BAT has a distinct dark color even at P0. In older pups, the BAT is surrounded by a thin superficial layer of WAT, which requires removal when the tissue is dissected. (C) Representative images of primary white and brown precursor cells after filtration through the 100 µm cell strainer, after the initial washes, and 24 h after isolation. Scale bars = 100 µm. Abbreviations: WAT = white adipose tissue; BAT = brown adipose tissue.

White and Brown Adipose Depot Collection from Mouse Pup: A Surgical Procedure to Harvest the White Adipose Tissue and Brown Adipose Tissue from Mouse Pup

Source: Galmozzi, A. et al. Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice. J. Vis. Exp. (2021)
In this video, ...

In mammals, adipose tissue or fat depot - a specialized connective tissue composed mainly of fat cells or adipocytes can be divided into two major types - white adipose tissue or WAT and brown adipose tissue or BAT.
The WAT consists of white adipocytes with few mitochondria and single lipid droplet, and stores energy. The major WATs are the subcutaneous inguinal WAT and visceral WAT.
The BAT comprises brown adipocytes with numerous mitochondria and lipid droplets and regulates body temperature. This tissue can be found in the interscapular, cervical, and axillary regions.
To isolate these adipose tissues, begin with a euthanized mouse pup in the left lateral position. Next, make an incision on the abdominal skin. Gently retract the skin towards the limbs.
Identify the subcutaneous WAT located within the innermost layer of skin, anterior to the upper segment of the hind limbs. Carefully excise the thin, elongated subcutaneous WAT from underneath the skin.
Now, position the mouse pup in the prone position. Incise the dorsal skin from the shoulder blade or the interscapular region towards the head. Locate the interscapular BAT - the deep red tissue between the shoulder blades. Gently remove the interscapular BAT.
Wash the harvested adipose tissues with a suitable chilled buffer. Store the adipose tissues in tubes containing buffer at low temperatures until further processing.

To collect subcutaneous white adipose tissue, cut the skin around the abdomen of a euthanized newborn pup, and gently pull the skin below the legs. Without collecting any skin tissue, carefully harvest the fat depot, which will appear as a clear or white, thin, elongated tissue attached to the inside of the skin or on top of the quadriceps. Rinse the harvested depot in PBS and place the adipose tissue in a 1.5 milliliter tube containing 250 microliters of PBS and 200 microliters of 2X isolation buffer on ice.
To collect interscapular brown adipose tissue, pull the skin from the shoulder blades over the head, and lift the deep red brown adipose tissue located between the shoulder blades. Carefully make incisions all around the adipose to detach it from the body, and check the tissue for consistency and color. After rinsing, place the tissue in a second 1.5 milliliter tube containing 250 microliters of PBS and 200 microliters of 2X isolation buffer on ice.

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4.6K Views. Źródło: Galmozzi, A. et al. Izolacja i różnicowanie pierwotnych białych i brązowych preadipocytów od nowonarodzonych myszy. J. Vis. Termin ważności (2021) W tym filmie pokazujemy procedurę chirurgiczną polegającą na izolowaniu podskórnej białej tkanki tłuszczowej i brązowej tkanki tłuszczowej między łopatkami od nowonarodzonej myszy. 

Video: Kolekcja White and Brown Adipose Depot od Mouse Pup: procedura chirurgiczna mająca na celu zebranie białej tkanki tłuszczowej i brązowej tkanki tłuszczowej od szczeniaka myszy - Experiment

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Identyfikacja i sekcja różnorodnych mysich magazynów tłuszczowych

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

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Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Izolacja i hodowla dojrzałych adipocytów ludzkich przy użyciu błonowych dojrzałych kultur zagregowanych adipocytów MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Izolacja i hodowla dojrzałych adipocytów ludzkich przy użyciu błonowych dojrzałych kultur zagregowanych adipocytów (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Wizualizacja i kwantyfikacja brunatnych i beżowych tkanek tłuszczowych u myszy przy użyciu obrazowania [18F]FDG Micro-PET/MR

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Wizualizacja i kwantyfikacja brązowych i beżowych tkanek tłuszczowych u myszy za pomocą obrazowania [18F]FDG Micro-PET/MR

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

White and Brown Adipose Depot Collection from Mouse Pup: A Surgical Procedure to Harvest the White Adipose Tissue and Brown Adipose Tissue from Mouse Pup

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