Dear Selma,

This does not exactly look like primary cortical neurons. Can you specify:

- from which species did you take them?

- how old were the embryos you made the culture with?

- do you have an image of the cells from the first few days of cultivation?

- why did you trypzinize them? and how did you do this exactly, as there should not be any cells left attached to the plate)

- do you cultivate other, stable neuronal cell lines in your lab?

Cheers, Laura

hello,, thanks for reply..

I used rat cortex of 18 days,, i always have a problem that cells clumps in neuroshere giant cells and large empty spaces in the plate with rolling cells seen by eye,,,and cells not make a confluent layer or differentiate into neurons,, .

.i was afraid of hypoxia and death of cells so i tried to dissociate them to dcrease number of cells so i did mechanical and trypsin dissociation,, most of cells died and the left made this shape,,

I have not work with cell line ,, this is my first time to try adjusting protocol::

I attached he photo of the first days ,, I have that big cells and spaces so i trypsined,,

Dear Selma,

By dissecting cortex from embryos you will always get a mix of neuronal and gial cells. The glial cells have the ability to proliferate, whereas the neurons does not have this capability. That means that the number of neurons that you have in your culture will never increase and will rather decrease if some are dying.

If you trypsinate the primary culture and seed them again at lower density then you will have fewer neurons and fewer glial cells to start with, but the glial cells will sart proliferating and after a few days you will have a much higher proportion of glial cells compare to neurons in your culture. I believe it is probably what you see on the picture after trypsination.

If you want to avoid the big clusters of neurons that you could see on the pictures a few days after dissection, then you need to be more careful with your dissociation step during the dissection, before seeding the cells for the first. Usually primary cultures of neurons are not trypsinated and they can live and grow for sevreal weeks after seeding.

Best, Maeva

I agree.  Primary neurons are plated and never passaged.  Either your did not obtain a single cell suspension from your isolation or your coating is the problem.

It appears that you have a nice pure monolayer culture of type I astrocytes.  Incidentally, this would make the perfect substrate on which to plate another round of primary dissociated neurons.  By culturing neurons on a feeder layer of astrocytes you will get faster and greater synapse formation and ultimately much healthier neurons, even at low density culture conditions.

hanks so much for reply,,I tried 0.1% PDL,, is beteer but still momolayer became like a roll,,  also still has the problem of neurospheres,, more clumps always,,

1)which cells number you plate per MWP6::(I plated 400.000 c/ well)

2) how i will be sure of neuron maturation?

also

3),, I need pure protein,, I found a protocol use ARA-C to stop the growth of glia,, is it ok

4)which best buffer to collect neuron protein,, and how,, directly using buffer or with scraping firstly

thanks

Hello Selma,

PDL should be fine. I like poly-ornithine a bit better. Leave the coating solution on at least overnight and wash the plate with sterile water before seeding the cells. 

Make sure that the cells are well dissociated (single cells and no clumps) before you seed them.

In order to have a high density for protein extraction, I would suggest you seed 1,000,000 cells per well of a 6-well plate.

You will see that your neurons grow and mature when they grow longer and longer neurites. You can fix them and immunostain for neuronal and glial markers to know the composition of your culture.

Ara-C can be used to inhibit glia cells.

For cell lysis, you can first detach the cells with trypsin, spin them down, discard the supernatant and add lysis buffer. Or you add PBS, scrape the cells off the surface, spin them down etc.

Best would be to find an experienced researcher to help you in your place. 

Good luck, Laura