i want to culture both adipose tissue as well as primary adipocyte but i heard its difficult to prevent it from floating
Hi Noura, adipose cells should be adherent. If they float, they are dead.
Try serum coated plates or Matrigel coated plates.
Mature adipocytes do float and tend to lyse over time but for short term experiments (7 days or less) you can use them as floating cells just fine. If you are differentiating them in vitro from preadipocytes then I coat the plates with collagen to keep them attached or use Corning Cellbind plasticware, both methods work. I have used isolated primary human adipocytes often and floating cultures are fine.
I concede to Eric who obviously has more experience in this field than I do.
Do you have a protocol for culturing adipose tissue not cells
from mice ?
I've maintained mouse and rat explant cultures in DMEM:F12 with 1%ITS+ and antibiotics for up to 10 days. Just make sure the explants are small (in the 1-3 mm range for all dimensions) and you have 5-10 explants per well in a 24 or 48 well plate. Conditioned medium can be harvested and replaced with fresh medium every 1-3 days. If what you are looking for is unstable the conditioned medium can be combined with serum containing medium or medium with BSA to stabilize it. Ive used this method numerous times to look at prolactin release from adipose tissue as well as various adipokines such as adiponectin and leptin.
Thanks a lot, i am collecting the media to isolate exosomes, i will try with your mentioned method but can i use 25 T Flask instead of well plate?
You should be able to but try to minimize sloshing the culture too much. I have always felt that the mature adipocytes interact with the polystyrene in a way that accelerates lysis.
Eric R Hugo Do you happen to have a protocol to coat plates with collagen to help the mature adipocytes adhere to perform co-culture experiments?
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