PBS kills neurons? - (Dec/13/2005 )

Hi everyone,

We are culturing primary mouse E14 neurons in Neurobasal medium with B27 supplement without an astrocyte feeding layer. The cells look really nice and healthy as long as we don't wash them with PBS. Just 10 minutes of PBS will actually kill them! Does anyone have the same experience? Is it a known phenomenon? I am thinking maybe the salt is toxic when there are no buffering astrocytes present? Any bright ideas?

Thanks!
Leonie

Hi

l cann't answer ur question...

But the saline in the PBS may work on neurons....

search saline activity on neurons in PBS...

Bye
Siva

Hi!

Can u see them after washing? PBS is without Ca...it´s needed for binding the ecm. perhaps u wash them away.You use porn/laminin dishes?

Greets Tobi

yes, they are still there after washing, but sorta look like they have disintegrated. You can still see their structure but they look a bit similar to after fixation with PFA or so. We do use PLL coated dishes and the neurons look great before washing. Raising the PH of the PBS did help a bit, but still they look not healthy at all..

Thx for the suggestion though!

try using KRH Buffer! its with ca...maybe this helps...and reducing time while washing...washing is just to get soluble factors and debris away. i wash only one dish a time!

We'll try that. Do you culture pure neurons or mixed cultures with astrocytes present?

adding FBS or BSA and Ca++ to any buffer helps to avoid stressing the cells. Anyway, primary neurons are very vulnerable, and advanced “physiological” buffers with supplements (i.e. glucose and BSA) should be used if possible. Also, do not dry the cells

probably the cells are not very happy after 10 mins in PBS. Simply rinse the cells with PBS, but not so long.

The same phenomenon happens with endothelial cells.

They can't bear PBS.

We use HBSS instead of PBS.

We use PBS-Sucrose for neurons in our lab. I don't remember off the top of my head what concentration we use. To be honest, I don't even know why we use PBS-sucrose for neurons. It is just one of those things I was told when I joined the lab.