This protocol allows the efficient and reproducible differentiation of brown adipocytes that express key adipogenic transcription factors, mature adipocyte markers, and have thermogenic potential and morphological phenotype of mature brown adipocytes. This is a simple and replicable two-phase differentiation procedure to obtain cells with characteristics of mature brown adipocytes from interscapular brown adipose tissue of newborn mice. This protocol allows studying the activation mechanisms of brown adipose tissue as a target for treating obesity.
It has been a model for determining the mechanisms involved in the pathophysiology of lipodystrophy. Performing this technique for the first time is expected to be challenging. It is essential to have a detailed protocol and emphasize critical points to have successful results.
To begin, place the euthanized mouse in a prone position and make a one centimeter long skin incision at the mid-level of the animal's back using sharp scissors. Remove the skin carefully, surgically detach the iBAT from the carcass using small scissors. Harvest both iBAT lobes using curve tip pliers to remove the lobes independently.
Place the two lobes in a plastic tube with 1.5 milliliters of sterile PBS at room temperature. Incubate the iBAT tissues in collagenase type two digestion buffer for 45 minutes at 37 degrees Celsius with continuous gentle shaking at 800 RPM. Mechanically disrupt the tissue suspension in collagenase type two digestion buffer by pipetting up and down using a P1000 micropipette and filter tips.
Use a new tip for each sample. Pass the desegregated tissues through individual 100 micrometer cell strainers into new 1.5 milliliter tubes. Add 500 microliters of ACK buffer to the filtered tissue samples.
Invert the tubes four to five times and incubate at room temperature for four minutes. Then pass the suspensions through a 40 micrometer cell strainer into new 1.5 milliliter tubes using a P1000 micropipette and filtered tips. Centrifusion resuspend the pellet as described in the text manuscript.
Then seed each sample into six wells of a 24 well culture plate by adding one milliliter of the suspension to each well. The undifferentiated preadipocytes presented very low or undetectable levels of ppar-gamma, C/ebp-alpha, perilipin 1 and CD36. In contrast, these markers were significantly higher on day seven of differentiation.
The accumulation of multiple lipid droplets in the brown preadipocytes was consistent with the previously published results. A significant increase was observed in the mitochondrial mass marker TOM 20 and the oxidative phosphorylation complex protein levels at day seven of differentiation. UCP1, a bonafide marker of this cell type was not detected in undifferentiated preadipocytes while it was substantially expressed in mature brown adipocytes at day seven of differentiation.
The mitochondria evolved from an elongated tubular shape at day zero toward a rounded or bean-like shape at day seven. The mitochondrial inner structure was also modified by adipogenesis, resulting in higher density of the parallel packed crystae. Importantly, the mitochondrial were intimately associated with lipid droplets in the differentiated brown adipocytes, so no discernible distance between the outer membrane of the mitochondria and the surfaces of the lipid droplets could be detected, even with high resolution transmission electron microscopy.
The surgical removal of interscapular adipose tissue is critical. An inefficient quantity of tissue limits the availability of cells that will be differentiated. This imaging technique helps characterize the morphology of differentiated brown adipocytes in vitro.
It's possible to perform assays for mitochondrial function, beta androgenic activation, and autophagic inhibition.
