Hello everyone,
I'm a phD student doing primary cortical neurons culture for 3 years. I'm doing this protocol from embryonic mice (E16) and following these steps
-Dissection in cold HBSS 1X +mg +Ca
-Cortices are kept in DMEM high glucose + glutamax supplemented with 10% FBS and 1% P/S
- After DMEM complemented is removed and trypsin 0.05% is add and cortices are incubated for 15 min at 37°C.
- Trypsin is removed and cortices are washed with DMEM complemented to stop the reaction, then dissociated in 1 mL with a P1000 (10-15 up and down) and filtered with a 40µM cell filter.
- Then we add 9mL of DMEM complemented to dilute cells and we count.
- Cells are plated in precoated PLL plates (100mg/L, washed 3 times with H2O) with DMEM complemented, 37°C 5% CO2.
- After 4 hours, medium is fully changed with Neurobasal + B27+ gentamycin + Glutamax
This protocol was working really well in our hands but since this summer is not working anymore.
We have a lot cell mortality even before plating the cells and the ones that are surviving after plating are presenting vacuoles and are stressed. We continue to see debris and mortality.
We changed with new solutions all our media, we autoclaved our instruments, we changed many "lot" of FBS. We tested another incubator, changed our coating. We did also a mycoplasma test which is negative after 2 weeks of culture.
During summer we had some changes in our cell culture facility (temperature changes, pressure, ventilation filter changes) and we are 4 users with the same problem (knowing that before summer for all of us, all was working well).
We notice that our media change color really fast (after less than one week after opening)
We changed I guess all and we don't have ideas anymore of what could be the problem.
Any ideas ?
Thx a lot
thank you Mahsa for your answer !
Like I said this protocol was working really well in our hands for 3 years and for many users. Suddently after the summer vacation (where we stopped our activity for 1 month) we started to have this problems.
We had tested directly in neurobasal, thinking that one of our media was the origin of the problem. But we have exactly the same thing. Even without filtration …
Other users joined our team after summer, and they were doing primary culture before. And now they have exactly the same unhealthy cells like us.
We tested each medium, each step one by one of course. And the results is always the same. We are thinking that something else changed in our cell culture box (which is a L2 facility) and this affect all neurons cultures. We excluded media or the protocol. But we don’t know what could be the point …
Thank you for your help
The main problem is the weather. You should perform experiments in a cold environment to retain the viability of the cells and also be quick in your protocol to minimize the time at room temperature since summer is not an apt season for culturing primary neurons
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