Yes unfortunately in my experience it does. The cell scraper with a disassociation solution was the best way to go. In fact we have found that trypsin activates all of the MAPK and even p53 and NF-kB.

Good luck

We had performed NGF differentiation experiments on PC12 cells without any problem. We used to plate the cells on collagen and poly-D-lysine precoated glass coverslips and cells were nicely distributed (no clumps). We used to dettach stocks without trypsin though (just flushing some media and resuspending cells by pippeting). You can find details in my J. Neurochem. paper (Herreros et al. 2000).

Can also be that different clones of PC12 behave differently.

Good luck

The proliferating PC12 cells tend to attach loosely to uncoated cell culture dishes. I recommend growing them on uncoated dishes and using only mechanical force to detach them (washing them with some medium by pipetting). Alternatively you can use 0.02% EDTA in PBS (Versene) if you have difficulties by using only medium.

For differentiation experiments I was plating the cells on polyornithine coated dishes and 24h later after washing them with medium without serum or PBS I was changing the medium to medium WITHOUT serum +NGF. I was using DMEM not PRIMI.

I hope this will help you.

Yes Judits suggestion of precoating plates may help.

1) Where did you get your PC12 cells from originally? If they were donated by someone else, are their cells performing as expected?

2) Is anyone else in the lab using these cells? Are they behaving the same as yours? Can you ask them to try their cells with your medium/coating conditions?

3) Do you know anyone else experienced with PC12 cells? Can you give them some cells and see if they get better results?

4) As Judit suggests, you cannot expect all PC12 cells to behave identically - they will have evolved over time, diverging from the PC12s used by other labs. Have you characterised your cells in any way? Can you compare karyotpye/genotype with other labs? Is there a PC12 'standard', that you can compare to?

5) Do you have multiple frozen 'batches' of cells (separately cultured flasks, frozen independently), or just a single batch? If multiple, perhaps a different 'batch' will perform better for you.

We routinely trypsinize our PC12s when passaging, however they only need a short treatment (10-20 secs) before inactivation. You might want to try lower concentrations of trypsin, maybe 0.025%..

I agree with Anton, keeping your cultures going on uncoated dishes is the best way to go. Also with passaging, make sure you collect ALL CELLS, floating and attached when splitting. When you are ready to differentiate, use laminin (or CAM of choice) coated flasks or slides. I have heard that if you don't passage your cultures properly (i.e. collect floating and trypsinised cells) you can select for a population that is less responsive to NGF. You might want to get some new stocks in (from a friendly lab!) just in case.

Kyle,

I did my PhD in the lab that did most of the early NGF-p75NTR/Trk work and characterization in PC12 cells so I can offer both advice and historical perspective. ANY TIME you take any cells out of the liquid nitrogen you should always passage them a few times to stabilize their properties and profiles before you use them. Though there are some exceptions to this, PC12 cells are not one.

Trypsin is a protease, which means in this context it goes and cleaves surface molecules and in doing so mimics a natural process cells use to signal a variety of physiological events. NGF application is a classic method for PC12 differentiation into a neuronal like state. As you eluded to, p75NTR signals via this proteolytic mechanism and p75NTR and Trk's are also the main component of the NGF signaling system. p75NTR, when it is cleaved, releases an intracellular domain which translocates and activates NFKB. So just thinking about NGF signaling you can quickly see how Trypsin can complicate the system's response in both inflammatory and differentiative responses.

PC12 cells should be routinely cultured in regular tissue culture dishes without coating molecules and should be dissociated with very little trypsin (if any at all) for a short amount of time. The cells are best passaged by blasting them off the plate with media (DMEM), sucking them up into your graduated pipette and essentially triturating them hard against the surface of the plate by sucking them up and spitting them out while the tip of your 5-10ml pippette is "firmly" planted against the surface of the plate. This is basically a physical means of breaking apart the little clusters they form into (ideally) single cells. Allow your newly split cells to grow overnight or longer, mostly to stabilize and adhere before doing any treatments. Use 100ng/ml NGF to differentiate for 5 days before use.

Don't worry what your cells look like (as long as they are healthy). Just count the total number of cells and the fraction of which bear neurites longer than 2.5 times the length of the soma. That's what'll give you an indication of if your treatment worked or not. People more routinely publish their best pictures that make the point they are selling as opposed to what was truly representative so looking at other folks pictures can be misguiding. Trust your numbers.

James

Hello Kyle,

I agree with James about the culture procedure for PC12 cells. In my experience culturing this cell line, sometimes is really hard to disaggregates the cell clumps before plating the cells and this is critical, especially for NGF differentiation. So I would suggest also, to use a syringe with 22g needle after doing the pipetting thing with the graduated pipette, and just before plating the cells. Best wishes

Laura

I always detach PC12 cells by pipetting up and down intensely and then I plate in poly-lysin coated wells (100 ug/mL overnight). You must dissociate the cells very well, avoiding cell clustering once plated. Also the density is critical and it can mitigate strongly the differentiation. Typically I plate them at 50000 per well in 24weel culture plates in DMEM 2% Horse Serum. After cells adhesion I add the NGF 50 ng/mL. NGF must be fresh for good neurodifferentiation and a new aliquot must be thawed each time. In such condition I get weel differentiated cells in 3-4 days

I suggest you should try using Collagen with Poly lysine or Laminin with Polylysine, as Trypsinization can cause damage on too long exposure but I don't think you would be doing that.

In my experience it has more to do with the clone of PC12 cells more than anything else. I tried PC12 cells from several different sources and they were all very different even in their ability to differentiate (most likely due to drift, the way they were handled/cultured-->different labs use different media formulations,plate coatings, etc.).

As an example a vial I got from ATCC never seemed to differentiate (it could have just been the one vial they gave me, never tried others), whereas PC12s I got from a former lab member frozen many years ago always differentiated robustly (and I would routinely trypsinize them in low % trypsin).

Thanks for all of the responses. The cell line I am using have been here since before I started working with them. I recently learned that the "strongly adherent" cells were selected for a while back. "The Expression of Nicotinic Acetylcholine Receptors by PC12 Cells Treated with NGF" by Rogers et al. mentions that they use trituration to "select against flat cells that adhere tightly to the dish and are not responsive to NGF". Some of you alluded to this. I am going to attempt to obtain some cells from a nearby University (as far as I know, I am the only one here working with them).

Thanks again for all the responses, I am definitely going to use some of the tips you all have given me!

In my postdoc I worked with a subclone of PC12 cells and performed many experiments differentiating PC12 cells with NGF. We used poly-D-lysine glass coated coverslips, then treated with rat tail collagen before plating the cells on glass. Trypsination did not affect the outgrowth of neurites.