Actually, I am trying to culture and measure hippocampal neurons from rat's puppies (E18) on MEA neurochip (256-9wellMEA300/30iR-ITO). With 30,000cell/well (~71,000cells/cm²) I could not seen any response on spontaneous transmitter release on two chips at DIV15 (with 468 electrodes in total).
Do you have any experience with that? Should I try 60,000 cells per well? Is it better to measure at DIV21 or later? I used to coat the wells with long-chain poly-L lysin (0.05mg/mL). As medium I am using Neurobasal + B27 + 0.5mM glutamine. How do you handle the fact, that almost 80% of the cells are astrocytes (according to NeuroProof GmbH, Rostock)? Are you able to minimize this, maybe by changing medium 1hour after plating the cell suspension? Are the astrocytes able to influence the hippocampal neurons responses?
Hello, I have been working with P0-2 hippocampal neurons from mice. I utilized the culture for patch clamp. I think DIV12-14 is reasonable. But to my experience hippocampal neurons are very sensitive to the preparation. The mechanical trituration has to be very gentle and you have to digest the tissue in enzymatic solution before trituration. Density I used was 300 cells/mm² on PDL coated coverslips. Add to your Neurobasalmedium 2% B27 and 1% Glutamax and exchange medium on the 2nd, 7th and 12th DIV.
Hi Dimitry, thanks for your answer. The reason why I am using more than the double amount of neurons per mm² is that the neurons have to grow over the electrodes by chance because I do not touch a single cell with a moveable electrode. They are fixed on the floor of that chips (26 electrodes per well).
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