Question
Asked 14th Jan, 2013
Could you tell me how I can transform raw information of the brainwaves in Hetz?
I'm using Neurosky technology, but the information about brainwaves are in raw. Then my goal is to transform raw information in Hertz, like the brainwaves theory says.
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How to depolarize cultured primary neurons with KCl without them dying?
Dear colleagues, I’ve started working with cultured primary neurons and came across a problem.
I need to depolarize neurons for different time intervals (up to 1 hour) and then use them for an assay 2 days after. The problem is that most of them are dying after depolarization.
I culture neurons in complete neurobasal medium (Neurobasal + 2% B27 supplement + 1%Glutamax +1% penstrep) with 1/3 media change every 3 days. I depolarize them on DIV11-14 by swapping media on Tyrode’s solution (45mM KCl) for up to an hour. Then I wash the cells with Tyrode’s solution (5mM KCl) twice and swap the collected media back.
45mM K+ Tyrode’s: 100 mM* NaCl; 45 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2; 1.04 mM Na2HPO4; 26.2 mM NaHCO3; 10.9 mM HEPES; 10 mM D-glucose
* NaCl is used to adjust osmolarity of the solution, so concentration varies.
Since in the current setup I depolarize cells in 5%CO2 incubator I used buffering formula of Neurobasal media. I adjust the pH to Neurobasal’s pH=7.7 and checked that in CO2 incubator it equilibrates to pH=7.4. And I adjust solution’s osmolarity to match the current neuron’s medium too.
Also, I depolarized neurons in live cell imaging using GCaMP6s to monitor calcium elevation and after minutes I can see that some neurites are destroyed (Fig.1, attached) and after 0.5-1 hour cells don’t look good and most of them die afterwards (Fig.2).
At the same time, I keep seeing papers with no explicit details on solution and osmolarity where cultured primary neurons are stimulated with KCl for hours. For example, here 6 hours of 55mM KCl (https://www.nature.com/articles/nature09033).
I guess there are a lot of people routinely working with primary neuronal culture. Could you please help me, what am I missing?
