hi guys,
my differentiated PC12 cells do not stick nicely to poly-d-lysine coated well plates. They are easy to wash out after a few washes by pbs or hbss solution. Any idea why and/or what to use instead? Could the adherence change while treating undifferentiated pc12 and incubating with NGF 100ng/ml?
Hello Marcela.
I found PC12 cells stuck well to polylysine, but they are notorious for phenotypic variability so your experience might be different. I also differentiated them polylysine/laminin, which gave much better neurite outgrowth. A cheaper and simpler alternative is Cell-Tak from Corning, although it´s obviously not very physiological. But then neither are PC12 cells!
Good luck!
Hi Sean,
thanks a lot for a prompt response. Please, would it be possible you send me your protocol for coating using poly-lysine and laminin? Also, what time do you use to treat the undifferentiated PC12 cells after the seeding? I usually treat them by NGF 100ng/ml 24 hrs following seeding, just when they are adhered. I would really appreciate any advice regarding this. Also, what do u mean by "phenotypic variability"? what is the difference btw different batches of PC12 cells? Thank you once again.
PS: Sean, do u use poly-L-lysine or poly-D-lysine? Just curious. I was told that could also matter.
Heh. We varied the coating protocol a lot as our main interest - primary adult DRG cultures - were very finicky. For them we used 10mg/ml poly-L-lysine (Sigma) overnight at 37oC, then 10mg/ml laminin (UBI) overnight at 4oC. You can use a lot less of both and get good adhesion with PC12 cells. I've also used poly-L-ornithine but never poly-D-lysine, but I have also heard that it can make a difference.
NGF at 50ng/ml 24 hours after seeding, cells used 3-7 days after NGF.
Phenotypic (and genotypic) drift is an underappreciated problem of using cell lines, particularly ones that have been around a long time and passed from lab to lab without checking. The process of culturing puts the cells under selective pressure with many variables, so the lines diverge over time in different places. Your PC12 cells are not necessarily the same as PC12s in another lab, even if you got them from the same source. This problem was known when I was a grad student 30 years ago and I occasionally see it mentioned in papers. The reason I mentioned it is that your variant might have poor differentiated adhesion characteristics. Do you have seed stock frozen down? Or you could get a vial from a lab where they don't have the problem.
thank you a lot Sean, for valuable info. Well, I have been passaging my PC12 for a while, so perhaps that is the reason. I will try some from frozen stock and see. I just started to work with differentiated PC12, thus I am not aware of all difficulties it may bring. One more question, what do you use to wash out the coating agent? PBS or sterile water? Once again, thank you for your input.
Best wishes, Marcela.
Sterile water for the poly-L-lysine then air dry in the hood. I´ve also air dried the polylysine before washing, but I can´t say that it was any better. The laminin was washed with cold sterile PBS, then once with warm culture medium before seeding.
Hi Marcela and Sean,
Nice to hear your practical solutions. I am getting very good response (Neurite outgrowth) with NGF 5O ng/mL. Serum starved (for 4 hrs) Cells were seeded after an hour coating with NGF and followed by single wash with sterile PBS.
But, the issue is cells are detaching from the surface after staining with cell membrane stain supplied by Molecular Probes, which I am not able to image under the microscope
Do you guys have any experience with this kind of work
Thank you
Raju
Hi Raju.
Many membrane probes are amphipathic and act like adding a small amount of detergent to your culture. You might be literally washing the cells off. Do they retract their neurites before coming off the dish?
Hi Raju, I have also found that the the cell viability stain form molecular probes to be absolutely useless for PC12 cells. It comes with their neurite outgrowth kit and the company told me that it definitely won't work on PC12 cells. hope you haven't wasted a lot of time on it as I have!
Has anyone tried growing PC12's adherently on poly D lysine coated flasks? It was recommended to me by the cell biologist at ECACC but I've never coated my own flasks but tried it out. The flasks too forever to dry in the hood and i wondered if it was ok to leave them to dry overnight without damaging the poly D lysine coating?
As long as your polylysine is sterile it shouldn't be problem. As for the flask question, you could try a stream of nitrogen through a pasteur pipette to speed up the drying. It should be sterile out of the cylinder, but make sure that you sterilize the tubing and/or put a filter before the pasteur.
- Badges
- Science method