Dear all,
I have harvested fibroblast cells from R26p-Fucci2 mouse embryos (mouse line as described in this paper: https://dev.biologists.org/content/140/1/237) and cultured them on plastic, hoping to obtain a batch of cells with fluorescence label indicative of their cell cycle state for further analysis. Before I harvested the fibroblasts and grew them up, I checked the endogenous fluorescence signals from the mouse embryos and I could clearly visualize them in the brain region, developing heart, lungs, etc. However, once I cultured them and passaged the fibroblast, the cells didn't seem to express the fluorescence markers anymore. I have also performed genotyping to confirm the presence of reporter genes in the mouse embryos.
The embryos were bred by crossing positive R26p-Fucci2 mice with WT C57 mice, and fluorescence signal were checked after the embryos were harvested from the mother. The embryos with fluorescence signals were processed as follows: First, the head and limbs of the embryos were removed. Then, the internal organs were removed by cutting the embryos open at the abdomen. All the internal organs including the kidneys, intestines, heart, etc were removed. The carcass of the embryos were then transferred to a new plate, and chopped to small pieces. The pieces were rinsed with 1x PBS and put in 1x TrypLE Express solution (approximately 5mL per embryo) in 4 degrees overnight. On the following day, excess TrypLE Express solution were removed where approximately 2 volumes of the solution were left with 1 volume of embryonic tissue. The mixture were incubated at 37 degrees for half and hour and the washed with 1x PBS afterwards. The cells were pelleted and cultured in DMEM (prepared following instruction from the manufacturer). Initially there were few fluorescence signal (around 10 cells out of 200 cells expressing reporters), but after passage, none expressed the reporter. Is there anything I've done wrong, or any procedures I've mistakenly performed that hindered the expression of the reporters?
Thanks for your time and help in advance.
Best regards,
Jordan
I don't know where you got this protocol, but I would directly start culturing the cells (without overnight incubation) after Trypsin or something like that.
Actually, TrypLE seems to be bad for cells. I would directly trypsinze them (about 1-2 hrs) and confirm fluorescence upon obtaining the cell culture in the dish.
At least, it worked in my case, I don't know.
Yulia Panina Thanks for your reply. It is a protocol recommended by the senior technician in my lab. The cells seems to be growing healthy and attached perfectly after the process, that's why I thought it is a valid protocol.
I definitely will try the way you recommended, thanks for sharing!
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