Daniel:Notebook/HiResChrPaint/2013-9-18
Fibroblast Adherence (Started 09/09/2013)[edit]
Given the bad results from Monday and Tuesday, I think it is time to put a little extra thought in to cell culture conditions. Did some research this morning on fibroblast culture conditions. I found an interesting [[1]] on UCSC Genome Browser that seems to be a simple (and useful) protocol for culture. The protocol recommends four ingredients in culture medium:
- DMEM (duh, provides all the essential nutrients)
- Fetal Bovine Serum (has a good deal of growth factors plus lots of BSA)
- 2-Mercaptoethanol (reduces disulfide bonds and acts as an antioxidant)
- L-glutamine (essential amino acid frequently added to culture)
I think we can skip the L-glutamine, and probably the 2-mercaptoethanol as well. However, I think the fetal bovine serum might help out the cell culture, and as it happens Song has recently ordered some, so I'll ask him about using it.
Update: talked to Alan. Apparently the medium we use already has other things spiked in it, and FBS is likely one of them, as well as L-glutamine (maybe not 2-mercaptoethanol since I can't smell it).
Another thing that was brought up was the high passage number. These cells may or may not be immortal, but they are on passage 14 or 15, and by now they're probably getting sluggish, whether or not they're immortal.
Further Evidence: I took a look today at the dishes, now incubated about 24 hours. Dish 1 and 2 looked dead still, but dish 3 was now highly confluent. Upon further thought, I think it is making more sense that the senescence is passage-caused. Dish 3 had the highest cell density, and therefore the greatest probability that at least some of the cells had not become senescent yet. Since dish 1 and 2 had low cell number, they were more likely to have no confluent cells and die off.