Hi everyone.
I am using PC12 cell line to check the effect of my interested gene on neurite outgrowth. My protocol as follow:
(1) Seed PC12 1x10^5 cells/well (12 well plate) and incubate 24hr with DMEM (high glucose) 10FBS 5HS.
(2) I used only 0.5ug of my interested gene/well and transfect using lipofectamine 2000.
(3) Leave the cells starve with DMEM 1% HS. Wells are pre-treated with Collagen type IC (sigma).
(4) Treat the cells with NGF 50ng/ml 0day, 1day, 3days, respectively.
However, PC12 started to die at day 1 after treated with NGF and cells died more badly at day3. Anyone who has experiences with this kind of experiments can help? Thank you so much in advance!
Thats an interesting culture effect.
I have used PC12 cells and differentiated them into neurites with NGF. They are generally not the easiest to cell line to work with but transfect OK with lipofectamine such as you have done here.
The following may be of help:
1.They do not like to be plated too sparse - but 1x10^5/well in 12-well should be ok. However, you might try and increase the plating density a little for transfection. If need be you could replate post-transfection prior to NGF treatment. You could transfect on Poly-lysine coated wells and then transfer to collagen coated as you like.
2. I suspect that your cells may not grow well on Type 1C collagen during serum starvation and NGF neurotic grow. The presence of serum aids cell attachment and its absence and differentiation may cause your cells to being poor adherent. If i remember correctly we used type IV (basement membrane) collagen for neurotic growth. You may want to play around with coating and concentrations for your particular assay.
I am actually having this exact same problem.
It is possible that the NGF is no longer viable and the cells die since they do not have a food source to stay alive since because of the serum deprived culture condition.
I would look back at the manufacturer's recommendation for reconstituting the NGF. Most of the manufacturers say to reconstitute in at least 0.1% BSA. Bovine Serum Albumin is a carrier protein that help keep the NGF stable, especially when you are diluting it to low concentrations. Although if you are reconstituting in FBS it should be okay...
Did you run a control without NGF?
Also DO NOT vortex the NGF.
Dear Kikuye Koyano:
Thank you so much for your recommendation! I had the control: PC12 without NGF. However, the cells have the same problem with the samples with NGF.
I also read some papers, other authors used the NGF with much higher concentration compared with my experiment (my exp. 50ng/ml). And I changed the medium every 3 days, 1 mL/well for 6-well plate. So I think the problem is not because of NGF.
Now, I have to accept that PC12 is really sensitive and easily detaches from the surface of dish. Therefore, now I try to be careful at the immune staining steps to observe the morphology of the remaining cells. I hope it will helpful.
ANW, thank you and everyone so much for the comments and suggestions! Hope to receive your suggestions next time!
Nguyen
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