The overall goal of this procedure is to isolate primary adipocytes and induce their differentiation in culture to beige or bright cells. The first step is to dissect adipose tissue from mice. Next, the tissue is digested with collagenase.
Once the stroma vascular fraction is isolated, the cells are plated. The final step is to induce differentiation of the cells to beige or bright cells. Ultimately, analytical methods are used to show increased expression of brown adipocyte specific genes.
This method can help address the key questions in brown adipose tissues, which is what molecular mechanism regulates the development of bright or be cells. The implications of this technique extend toward treatment of obesity and other lifestyle diseases. Since the manipulation of brown adipose tissue in humans can be used as a potential therapeutic intervention, The first step is to prepare the digestion medium.
Then after euthanasia, the adipose tissue is removed. First, isolate the interscapular brown adipose tissue or bat by cutting along the back of the animal all the way to the neck. Two lobes of brown adipose tissue can be found right under the skin between the shoulders.
Next, carefully remove the thin layer of white fat covering the bat. Once isolated, place the tissue directly into clean PBS. The inguinal white adipose tissue or wat is found directly under the skin on both sides.
It extends to the inside of the thighs and down toward the testicles. Remove the tissue and place in clean PBS. The following steps are applied to both the bat and wet samples.
Quickly remove all contamination such as hair, skeletal muscle, and connective tissue from the tissue sections. Next, dry the tissue quickly on a paper towel to prevent the dilution of dissection medium with PBS and place it on a dry plate. Add the digestion medium and mince the tissue into small pieces.
Next, transfer the tissue into a 50 milliliter tube containing the rest of the digestion medium. Place the tube in a 37 degree Celsius incubator with constant agitation for 40 to 50 minutes to digest. Check every 10 to 15 minutes to make sure the digestion is going well and to prevent over digestion.
After the tissue is well digested, stop the digestion. By adding five milliliters of complete medium and mix well, the cells should be almost completely homogenous. Next, centrifuge the sample at 700 times G for 10 minutes.
The stromal vascular fraction should now be visible as a brownish pellet on the bottom of the tube aspirate the oily mature adipocyte layer on top and most of the liquid layer. Next, dissolve the pellet in 10 milliliters, complete medium and mix well. Once mixed, place a cell strainer over a new 50 milliliter tube and filter the cell suspension.
Transfer the filtered cell suspension to a 15 milliliter tube and centrifuge at 700 times G for 10 minutes. Next, aspirate the medium and resuspend the pellet. In 10 milliliters of complete medium pipette and mix well plate the cells as needed.
Generally, five mice will yield two 10 centimeter plates of inguinal wat and one plate of bat. One or two hours after plating. Aspirate the medium wash with PBS twice and add fresh medium.
This step is important as this can remove red blood cells, immune cells, and other contaminants. The maintenance and induction mediums are prepared ahead of time. Once the primary adipocytes have grown to 95 to 97%confluence, change the regular complete medium with induction medium.
After 48 hours, change the medium to maintenance medium containing 0.5 micromolar RO glitazone. After an additional 48 hours, change to fresh maintenance medium containing one micromolar RO glitazone for additional two to three days. Droplets will appear around three to four days after the induction as seen here six to seven days after first adding the induction medium.
The cells should be fully differentiated to mature fat cells and should be filled with oil droplets. Cells can now be harvested and used for further analysis. Browning of primary adipocytes can be assessed by measuring mRNA of brown fat specific or selective genes by Q-R-T-P-C-R as seen here.
RO glitazone, robustly induced UCP one mRNA expression FORSKOLIN treatment designated here as cyclic A MP.Further augmented the RO glitazone induced UCP one expression.Cdia. Another brown fat selective gene was also robustly increased in a dose-dependent manner. RO glitazone treatment also increased the expression of FABP four and adipogenic marker for both brown and white fat.
This browning effect was not simply due to an enhancement of adipogenesis per se. Since the induction of UCP one and CDIA expression was still significant, even if the mRNA levels were normalized to those of FABP four, this Western blot confirms the increased protein level of UCP one. After watching this video, you should have a good understanding of how to isolate preadipocytes and how to differentiate these cells into beige or bright cells in culture.
