My cells are becoming confluent near the walls of single well plate, but population is sparse at the centre. Not able to synchronize them before inducing differentiation.
Dear Sushri,
These cells are not immortalized and you have to use them at lower passage. They are suitable for only 12-13 passages. Also, you have to be sure that cells are 100% confluent for 48 hours prior to initiating differentiation. For that, seed them at a density of 50000 cels/mL in 24 well plates using DMEM supplemented with 10% calf serum in order to reach confluency. Do not use fetal bovine serum during the proliferation process. It will affect later differentiation potential.
Dear Sushri,
These cells are not immortalized and you have to use them at lower passage. They are suitable for only 12-13 passages. Also, you have to be sure that cells are 100% confluent for 48 hours prior to initiating differentiation. For that, seed them at a density of 50000 cels/mL in 24 well plates using DMEM supplemented with 10% calf serum in order to reach confluency. Do not use fetal bovine serum during the proliferation process. It will affect later differentiation potential.
I find it helps to shake the plate in a linear motion in two perpendicular directions. Are you swirling the cells in a circular motion? I've found that can thrown the cells to the outside before they have a chance to settle and attach to the plate.
Thanks for the response. I have few more issues with these cells. I use FCS for proliferation and FBS for differentiation and maintenance of these cells. However, I noticed that a few days after inducing differentiation these cells have started detaching from the plates and start floating. These are loaded with lipid droplets even in floating condition. Is it a normal thing with these cells or do I have to handle them differently. I have followed the guidelines provided in this link
http://www.atcc.org/attachments/22129.pdf
and the single well plates used were from Hi-media and were meant for culture of adherent cells.
Have you tried using FBS for proliferation and differentiation? Are you changing any other ingredients in the media or are you simply adding the proper concentration of differentiation inducing chemicals to the normal media?
For preadipocyte proliferation I used FCS, but have never used FBS for proliferation. I am using FBS only for differentiation and maintenance. Could this be because of the passage number.
I've been able to keep using the cells as late as passage 20 without problems. After that and it becomes a bit unpredictable. Is it possible you're getting trypsin mixed in with your media?
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