Hi,

undifferentiated PC12 are likely to grow in 'suspension clusters' so if you want to fix them for further imaging you may try to work in microtubes and process them like staining cells for flow cytometry (collect, spin, fix, wash, spin, stain, wash, spin...) and eventually lay them on glass slide for microscopic observations. NGF-dIfferentiated PC12 should adhere and grow neurites-like extensions on a poly-lysine/laminin coating, you can find relevant concentrations and protocols in litterature. lamini is very fragile I would advise to use -80°C freshly (less than 2 months) frozen aliquots.

hope it helps

thank you for your advice but unfortunately it doesn't apply to what i am attempting to achieve.

I'm attempting to treat the cells with a DRC of NGF on a 96 well plate. Which works beautifully and you can see by eye the difference in neurite outgrowth down the plate, but i was attempting to make the quantification less laborious by staining the microtubules, but as soon as you do a wash step you lose the majority of undifferentiated cells, short of counting every cell in a well manually and assessing the neurites myself with neurite tracing software, I'm at a loss as how to fix this. 

Hi, maybe yould try to quantify cellular extension growth simply (without any staining) by using ImageJ software, very usefull for this kind of application.

Yeah I was trying to find a less laborious alternative but it is looking like my only option at present. thank you!

I know I am a little late to the party, but in my experience of culture PC12 cells and numerous types of primary neural cell cultures. the issue is two fold, 1 - is the attachment of your ECM to the culture surface (we coat with PLL and let dry overnight, followed by laminin for 2 hours immediately prior to plating the cells). and 2 - when PC12 cells are partially differentiated they don't stay stuck and this is primarily due to incomplete formation of the junctional complexes with the ECM (they will stick very poorly to PLL alone)

if you are culturing and imaging the PC12 cells directly on the TCP of a well plate or petri dish, a good trick is to

Remove the majority of the culture medium

Add approximately an equivalent volume of 2x the conc of formaldehyde you require.

Allow to fix for 10 minutes

Remove approx half of media with formaldehyde

Add 2% agarose (low melting temp is best, dissolved in PBS) making sure the agarose is just above it's setting temp when you add it so as not to kill the cells.

Once agarose is set proceed with washing and staining the culture (you can put on ice to decrease setting time if required)

Hi Aaron!

Thank you for your reply always appreciate multiple ways of of doing things! I have never heard of using agarose - very neat trick!

I had previously tried poly L and laminin, but as you said, when they're differentiated they tend to fall off! I found that I was still losing quite a few cells when only partially removing media and unfortunately this wasn't consistent enough for a quantitative assay that I was trying to achieve. However, definitely suitable if you're just looking to visualise an effect on individual cells.

I eventually solved the issue by passing the cells through a syringe (3 times) to declump the cells and then using Corning's Collagen IV at 1ug/cm2 to coat 96 well plates. I'd previously steered away from collagen as my colleague (previously set this project) had tried other brands and couldn't get them to work and it was my final hope (I'd exhausted ever other option I could find at the time) and it worked a treat! Would highly recommend to try if you're to do any future work with them.

Thanks again!

Rose