Adherent cells not adhering - (Dec/28/2006 )
I'm working on my final yr project. The topic is- "Characterization of Heat shock proteins of mammalian cells."
Funny thing is, our project superisor insisted that we use this cell line -CHO (a recombinant strain that produces interferon gamma; adherent cell line)
We're investigating HSP 47 and 27.
Last friday, (22nd dec), I thawed a vial of cryopreserved cells. Normal thawing procedures were obeserved. The cells looked fine under the microscope.
Today, however, the cells have yet to attach to the surface of the flask (T25). The cells look bright and round (like normal healthy cells) except that they're not adhering to the flask surface.
We've tried to introduce thermal shock to the cells at 42 degrees Celsius for about 5min, and collected the cell samples. WE wanted to extract proteins (primarily HSP 27 and 47) for further analysis. We lysed the cells using a special combination of lysis buffer and sonication. Thereafter, we ran SDS-PAGE gels for Coomassie blue staining for detection and Western blotting.
No results obtained for western blotting though.
WE're speculating whether or not to carry out 2D gel electrophoresis.
Since the topic is on Characterization of HSP for our cell line (which happens to be CHO which is WELL CHARACTERIZED). Besides, there are little science journals found on HSP 47. I have no idea how to go about carrying out experiments for further analysis other than detection methods. Any suggestions?
It sounds like this line has been adopted for suspension culture. What type of medium are you using to grow them? I would suggest you optimize western blot condition by testing different antibody sources and changing antibody titer, detection method etc. Other alternative will be to use ELISA.
Thank you for your suggestion. We're trying ELISA now. About changing antibody sources, we aren't able to do just that. We're given a primary antibody anti-mouse HSP 27 and anti-mouse HSP 47.
Secondary antibody is mouse anti-mouse antibody coupled with HRP. I believe the primary antibody should be specific to our cell line we're using.
We grow the cell culture in MEM medium with a minute concentration of methotrexate to inhibit excessive growth. The cell line is adherent as I've been culturing it for a month now. There was a contamination earlier on so we had to thaw new vials. I was just wondering why didnt the newly thawed cells attach to the flask surface (though they might be looking v much alive under the microscope.).
Do you think they're dead already?
it is one of good project (charecterization of HSP).Since Iam working on cho cells for 4 years, I observed that effect of Hsp on recombinant CHO CELL LINES really increase the cell viability and immortalility of cells, thereby increases the yield in bioreactor.
So it is better to do the project on chareceterization of HSP and its EFFECT ON PRODUCTIVITY AND SCENESCENES.
BEST OF LUCK...,