Functional study with PC12 cells - (Jun/06/2006 )

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QUOTE (molecule @ Sep 13 2006, 10:14 PM)

where can i buy this antibody from? have you use it before?

we use tau and it stains perfectly well. we didnt buy it. the PI got it from his old lab.

I remembered the name of the other antibody used for dendritic detection- MAP2

U could look in the literature for some papers using these antibodies and check the company as well.

-scolix-

QUOTE (scolix @ Sep 14 2006, 10:14 PM)

QUOTE (molecule @ Sep 13 2006, 10:14 PM)

where can i buy this antibody from? have you use it before?

hi by the way, do you grow your PC12 on collagen coated flask?
I have difficulty getting all cells detaching with trypsin, in the i need to scrape.

How long can I expose the cells to trypsin? does scraping recommended during passaging step?

-molecule-

QUOTE (molecule @ Sep 19 2006, 09:56 PM)

we dont use PC12 but SHSY5Y and we grow them in the usual flasks. But when we want to differentiate them , we grow them in plates coated with Lysine and Matrigel.

I incubate my cells with trypsin for 5-7 min. while passaging and not scrape them.

I dont know how its for PC12.

-scolix-

QUOTE (Kathy @ Aug 31 2006, 01:44 PM)

i also use PC-12 and coat plates with collagen, i dont remove it, keep it there for the whole study. pour in solution carefully, adhering pipet to the wall not directly onto the cells.

hi kathy,

need ur advice here. I am working on pC12 too. and i coated my flask with collagen.
however I have difficulty getting majority of cells rounded up upon trypsin treatment up to 5mins. In the end I have to scrape them.

1) How long can I expose the cells to trypsin?
2) Is scraping ok when doing passaging/subculture?

-molecule-

QUOTE (jmo @ Aug 30 2006, 12:47 AM)

QUOTE (Fan @ Jun 6 2006, 07:46 AM)

Hi, I am currently doing Ca2+ influx study with PC12 cells (culture for 6 days with NGF). Since this cell line is not strongly attched, then the cell total no. could not be controlled after several wash, so anyone has done or been doing this kind of experiment, could you please give me some suggestion? Thanks

Hi Fan, I also use PC12 cells for functional studies. I coat my plate with 1mg/ml Pol-L-lysine and rat tail collagen before transfecting my cells. I also treat them with NGF for 3-5days before I proceed with immunocytochemistry or Immunoprecipitation. I have not have any problems. If need protocol for plate coating let me know.

Hi jmo,
I soon be using NGF to differentiate my PC12 cells.may i know what you get the NFG from? the working concentration?

What antibody can I use to see the differentiated cells under fluorescence microscopy or western blot assay?

May I request for your protocols on immunocytochem and immunoppt and collagen coating ?

One last time, I have difficulty rounding up the cells upon trypsin treatment, in the end i need to scrape to gett all cells detached. Did u face this problem? how much trypsin u add for 75cm2 flask normally, for how long duration?

thanks

-molecule-

QUOTE (rocky raccoon @ Jun 9 2006, 06:37 AM)

QUOTE (Fan @ Jun 6 2006, 07:46 AM)

Hey man, well I was also having the same problem my PC12 cells were not sitting down, do this coat your plates with collagen solution (35ng/ml) and let the plates sit at 37 C for 15mins remover collagen (you can reuse this for later) wash the plates with PBS and then plate your cells on it, they will stick like anything...hope this helps

hi rocky

I have difficulty getting majority of cells rounded up upon trysin treatment. The cells seem to stick so strongly on the plate coated with collagen? how

-molecule-

Hi. We're trying to transfect and image PC12 cells. Does anyone have a good protocol for coating coverslips with collagen IV? And would you transfect before or after adhering the cells? Thanks!

-katherine_rockefelleruniversity-

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