I am trying to isolate the mature adipocytes directly from chicken abdominal adipose tissue, ceiling culture the adipocytes, and then isolate the RNA from these proliferated adipocytes. I followed the protocols from several published articles, but none of them work well on these chicken adipocytes. So I am wondering if it is a better option to induce the adipocytes from pre-adipocytes, and then extract RNA from these cells. My doubt is whether such fresh mature adipocytes isolated directly from adipose tissue will give the same gene expression results as those adipocytes induced from pre-adipocytes. Because the directly acquired adipocytes are actually induced in vivo. Though by adding hormones, the pre-adipocytes can also be induced to adipocytes and firmly attached to the culture dish, it is still not a precise mimic of the in vivo environment. So does anyone have any experience if these two sources of adipocytes give different gene expression results? (I am interested in the genes involved in proliferation and differentiation)
The cells derived from the adipose tissues contains a large fat storage (looking eccentric nucleus). 3T3-L1 preadipocytes look like many other non-adipocytes even long after induction. They then begin to show many small fat globules within the cells, eventually making much larger fat storages.
Yes, absolutely as you said. If your are interested in the genes involved in cell proliferation and differentiation. it would be O.K. The only worry is that in vivo and in vitro are so different. and not all cells differentiate into one direction by chemical induction in defined medium.
You'd better compare preadipocytes from juvenile chicken and adipocytes from adult abdominal cavity.
Thank you for your suggestion Dr. Lee! I agree that even the isolation procedure is carefully conducted, the multipotent MSC/ADSC can still be stimulated into different lineages within the same culture condition. Also when I isolated the adipocytes, even the cells were washed carefully for several times and the ceiling culture procedure was used due to the floating nature of the adipocytes, the adipocytes can still be contaminated by other cell types which were not supposed to be floating on top and attach to the ceiling. So even now I got cells attached to the ceiling, the morphology showed that these are preadipocytes but not mature adipocytes. So it might be really difficult to isolate and culture pure adipocytes out of other cell types. I am trying to figure out if by using flow cytometry, I could sort out the adipocytes by using specific markers so that the following treatment can be given then to the pure adipocytes.
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