To investigate the biology aspects of brown adipose tissue, it is essential to clear purified brown adipose size. Currently no came in brown adipose size purification and the masses are available. In this study, we developed a straightforward protocol to address this issue.
Now, one skills are required for this protocol. It needs much fewer studying materials than other methods and the purified brown adipose size can be directly used for gene and protein expression analysis. This protocol provides a new method for studying the biology of classical brown adipose size.
It can also be applied to study white adipocytes browning process. Demonstrating the procedure will be Amanda, a research assistant from my laboratory. Start by placing the flask with BAT and digestion solution on a magnetic stirrer at 60 rotations per minute in a 35 degree Celsius incubator for 30 minutes.
Use a one milliliter pipette to disrupt the aggregated tissue clumps of BAT slices around the stirrer bar. After digestion, place a 70 micrometer strainer filter on top of a clean 50 milliliter centrifuge tube and pipette around four milliliters of cell suspension through the strainer. Then wash it with four milliliters of 12%iodixanol solution.
Pipette up and down to mix the cells with iodixanol solution then transfer the cell mixture into two clear five milliliter polystyrene test tubes. Place the polystyrene tubes containing the cell mixture on ice for one hour, causing the BAs to form a layer on the top. Take out 20 microliters of the isolated BAs for microscope examination.
For RNA and protein isolation, pipette the BA layer into two 1.7 milliliter microcenterfuge tubes. Then carefully remove the excessive iodixonal solution without disrupting the BA layer. Then add one milliliter of TRIzol into the cell solution to simultaneously isolate RNA, DNA, and protein and mix sufficiently to lyce the cells.
To separate the phases, add 200 microliters of chloroform and centrifuge to the tube at 9, 981 G for 10 minutes at four degrees Celsius. After centrifugation, use the aqueous phase for RNA isolation. Transfer 300 microliters of the organic phase into a two milliliter microcentrifuge tube and add 750 microliters of 100%ethanol.
After vortexing for 10 seconds, add 200 microliters of 1-Bromo-3-chloropropane and vortex again for 10 seconds. Add 600 microliters of double distilled water before vortexing for 10 seconds. Let the mixed solution stand for 10 minutes at room temperature.
After centrifugation, add 9, 981 G for 10 minutes at four degrees Celsius. The phases will be separated with the protein phase localized in the middle layer. Remove the top aqueous solution and add one milliliter of 100%ethanol into the remaining solution.
Following centrifugation at 9, 981 G for 10 minutes at four degrees Celsius, discard the supernatant and wash the pellet with one milliliter of 100%ethanol. Centrifuge at 9, 981 G for 10 minutes at four degrees Celsius. After removing the supernatant, air dry the pellet for 10 minutes at room temperature and measure the weight of the wet pellet.
Add 1%SDS solution at a ratio of 20 microliters per milligram of pellet. Dissolve the pellet completely by putting the tube in a heated shaker at 55 degrees Celsius and 11 G for five to 10 minutes. In the protocol, PBS containing 3%BSA was used to separate BAs from BAT.
The isolated cells were raspberry shaped with multilocular lipid droplets inside and were tdTom positive, confirming that they were BAs. Protein was isolated from the BAs with an improved GTPC protocol and was then examined with an SDS-PAGE gel. A dominant band associated with BSA was observed in the BA sample, suggesting its interference in protein extraction.
To avoid BSA interference, a 6%iodixonal solution was used for the isolation of BAs, which had a typical raspberry shape and contained multilocular lipid droplets. Proteins extracted from these BAs were well separated in the SDS-PAGE gel. These cells were found to be tdTomato positive whereas cells recovered from the pellet were tdTomato negative with no obvious lipid droplets, suggesting efficient separation of the BAs from the non-fat cells.
In gene expression analysis, the mRNA levels of UCP1 and PDGFA were significantly higher in the isolated BAs. But the mRNA of PDGFRA was only detected in BAT. UCP1 protein was found enriched in isolated BAs.
EDGFR alpha was detected in BAT, but not in isolated BAs. These results confirm the efficiency of the method for isolating BAs and demonstrate that the isolated BAs are suitable for gene and protein expression studies. When attempting this protocol, use fresh digestion buffer to dissociate brown adipose tissue and avoid tissue clump formation during the digestion period.
This new method makes it easy to investigate the biology of brown adipose tissue on a single cell tap level. By applying this method, one can perform GN and protein expression studies with pure brown adipocytes precisely dissecting brown adipocytes gene expression program, without worrying about contamination originally from nonfat cells.
