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NGF and PC12


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#1 iamnewhere

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Posted 28 June 2011 - 01:41 AM

Hi there..

Good day.. :) I will be working on differentiated PC12 cell. Normally people will use Nerve Growth Factor to differentiate the cell.

Some of people differentiated the cell in flask for 6-7 day and they will add or change NGF solution every 48 hr. After cells confluence about 70-80%, they will trypsinize/scrap and transfer to a new plate eg:24 well or 96 well plate for assay.

But others will plate the cell directly to the plate with desired cell density and differentiate the cell with NGF for 6-7 day and they will add or change NGF solution every 48 hr. Then, same as above, they will trypsinize/scrap the cell and transfer the cell into a new plate for their assay.

My question is:-
1) Does trypsin may effect the differentiation of the cell? Is it still diffrentiated after we trypsinize them?
2) Can differentiated cell change back to undifferentiated cell if we sub and grow it into a new flask? If yes, can we add NGF to differentiate the cell?

Did you know any link or people that good in this field?

Thanks alot for your concern. Hope to get reply from you soon.. :) ... Thanks alot.

Best Regards
-me-

#2 gamizz

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Posted 28 June 2011 - 10:45 PM

Hi,


I am working with PC12 cells almost 2 years and I never tripsinize them. According to the literature ( regulation of the differentiation of PC12 pheocytochroma cells,1989)tripsinisation deplete fast NGF receptors therefore it can effect cellular response to NGF. Therefore I always do pipeting to detach them. But I never touch the cells after differentiation, i mean I don't passage them. Because if you passage after differentiation you will harm neurites.

I hope it will be useful



Hi there..

Good day.. :) I will be working on differentiated PC12 cell. Normally people will use Nerve Growth Factor to differentiate the cell.

Some of people differentiated the cell in flask for 6-7 day and they will add or change NGF solution every 48 hr. After cells confluence about 70-80%, they will trypsinize/scrap and transfer to a new plate eg:24 well or 96 well plate for assay.

But others will plate the cell directly to the plate with desired cell density and differentiate the cell with NGF for 6-7 day and they will add or change NGF solution every 48 hr. Then, same as above, they will trypsinize/scrap the cell and transfer the cell into a new plate for their assay.

My question is:-
1) Does trypsin may effect the differentiation of the cell? Is it still diffrentiated after we trypsinize them?
2) Can differentiated cell change back to undifferentiated cell if we sub and grow it into a new flask? If yes, can we add NGF to differentiate the cell?

Did you know any link or people that good in this field?

Thanks alot for your concern. Hope to get reply from you soon.. :) ... Thanks alot.

Best Regards
-me-
[/quote]

#3 iamnewhere

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Posted 21 July 2011 - 07:45 PM

Hi gamizz.

Thanks alot for your advice.. I just started my labwork on differentiating the cell.

I've read some journal and article about differentiated PC12 . It stated that Once we add the media with NGF, the cell will stop dividing and they will start produce neurite.

I have differentiating the PC12 cell for almost 4 day with 100ng/ml concentration. But it did not produce neurite and keep on dividing and almost confluence.

Im using PBS containing 1%BSA in preparing the 7s NGF and found that the NGF is not fully dissolve. I tried to vortex the solution and it still not dissolve. Is it the cause of my problem of neurite outgrowth?

Does the concentration of serum(eg: FBS, HS,BSA, FCS, etc) that we use in media may affect the reaction of NGF? Do we have to reduce the serum?

Hope to get reply from you soon.. Thanks alot.

Regards
-me-




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