HI Alex!

Let me start by saying I have never worked with any of those lines and I have definitively worked differentiating.

That said, I have been working for the last year with 2 fish cell lines, due to reasons that not important for the conversation I don't have access to a CO2 incubator. I spent easily 2 months testing ALL kinds of media. I can tell you right now L-15 saved my life. It is an extremely stable medium in absence of CO2 because it's chemically formulated to be buffered in absence of HEPES, NaHCO3 and CO2. Actually one of the cell lines (ZFL) is a very picky one based on the ATCC, and I am using just L-15 with FBS and they are happy!

You may wanna give it a try, if you haven't yet :)

Good luck

Nuria

Hi Nuria !

Thank you for your advice ! I'll definitely take a look at the L-15 medium.

By the way, as you mention ATCC, I would suggest people that want to work with PC-12 to be carefull with the one provided by ATCC. I worked several months on the ATCC cell line "PC-12 Adh" (Adherent phenotype), and tested a HUGE amount of conditions (serum, seeding density, pre-treatment/post-treatment timing, [NGF]...).

I never got any sign of a beginning of a neurite outgrowth...

Best regards

Alex

After some tests, differentiation works fine afer 24H if I culture the cells one day in a CO2 incubator in Low Serum, before placing them in the "non-CO2" incubator with the [Hepes / no NaHCO3] medium supplemented in NGF.

I think it's impossible to get anything good from a direct seeding in differentiating medium without a CO2 incubator. Cells seems to need CO2/NaHCO3 buffering for the settling phase. Sadly, because otherwise it would be a great gain of time...

Alex

Can you provide the complete composition of the culture medium (having HEPES)? HEPES come in two variation (sodium salt and ???) each providing different pH; a combination of both helps. Further, the pH of the medium changes upon addition of suppliments such as beta-ME and NEAA.

Hi Alexis,

I've recently been given a project in work, working with PC12 cells and differentiating them. Ive been using ATCC's cell line and i've been failing. Im glad now seeing your post that I'm not the only one exhausting every option. 

Just wondering if you've used Sigma's cell line and have a protocol for differentiation using NGF? I've wasted so much time with these cells already. 

Thanks

Rose

Hi Rose,

For my experiments, I used Neuroscreen-1 cell line from Thermo. It's an adherent PC12 subclone that responds well to NGF, and that makes no clusters in culture. However, I think that Thermo does not sell it anymore.

Before, I tried to differentiate the PC12 ATCC-Adh cell line, without success. 

I did not work with the classical PC12 ATCC cell line, and can't help you with this one.

Here is my protocol for the NS-1 cell line:

I seed my cells in high serum (15%) and let them grow for 24h. Then, I lower the serum for 24h (1,5%), and finally I treat the cells with 200 ng/mL NGF (Sigma) for 24 h, still in low serum. Treatment during 24 h gave me a significant and clearly observable neurite outgrowth. However, 48h/72 h treatment is optimum.

You can read the M&M of my publication in Neuroscience Letters for more details.

Hope it helps !

Alexis

Dear Alexis, 

I just started PC12 differentiation. They do not respond unfortunately. I treat them with 100 ng/ml of NGF. I am so confused now. I am wondering is it OK with you to share your protocol for pc12 differentiation. 

Dear Sahar,

Unfortunately the efficiency of differentiation will highly depend on what PC12 subclone you have. My Thermo Fisher NS-1 subclone was very sensitive to NGF. You can check any of my publications for the detailed protocol of differentiation I used.

Hope it helps!

Alexis

Hi Alexis

I have started to work on PC12 differentiation for about 3 months now.

I am using PC12 ADH from ATCC.

my protocol is below:

culture the cells in T75 collagen coated rat tail type , using DMEM 10 % FBS, 5 % horse serum. media is changed every other day and sub culture upon 70-80 % confluence.

for experiments, my cells are cultured onto polymeric scaffold using the same media above. one day after seeding, the media is changed to using dmem, 1 % horse serum, and 100 ng/ml NGF is added. the media are changed every other day over 5 days (i.e. NGF treatment over 5 days).

however, i have not managed to get much cells to differentiated. a few cells would differentiate. but even so, the differentiation is only partial.(the filament does not extend very long neurite, only short neurite).

I have read some papers, some used dexamethsaone, retinoic acid, cAMP and N2 supplement.

do you think you could share some advice? is it really necessary to add those other additional supplements like dexamethasone or retinoic acid or cAMP? why is it that some manage to differentiate with just NGF alone.

I am not sure how to proceed from here. I have heard cell density would affect. I have tried that too but still neurite is short.

Thanks!

oh, I should add that I did not collagen coat my scaffold although for maintenance, I will coat the T75 flasks with collagen rat tail type 1. not sure if this could be the reason for the failure to differentiate.

Dear Srah,

As I said in other answers to questions related to PC12 differentiation, that you can easily find, I found that PC12-Adh were not able to differentiate, whatever treatments and culture conditions I used. I would suggest that you use another PC12 cell line for neurite outgrowth assays.

Good luck !

Alex

Hi Alexis

I'm working on pc12 suspension cells and I've not differentiate them yet.

my question is about differentiating procedures.first , i should culture the cells on collagen iv coated plate and isolate the adherent cells and then add the NGF to differentiate cells to neurite shape?or at the same time culture cells on collagen coated plates containing NGF?

Thank you

Dear Shirin,

I did not work with PC12 cells grown in suspension, so I can't tell you what's best.

I'd just say that you should definitely try both methods, but from what I know, most labs are seeding cells in normal medium, and then treat with NGF in low serum medium at least 8-24h after seeding. I assume it's to let the cells "settle" in good conditions, before putting them through a stressful treatment.

best,

alex

Hi Alexis

Thank you for your answer .According the related articles that I've studied until now your description seems more reasonable.

Its very helpful.

Thank you

shirin