Dear all,
I was wondering how people add AraC to primary neurons in cell culture. I do a pre-dilution in H2O to achieve a concetration of 250 µM and add 5.6 µL of this solution to 500 µL medium in a well in a 24-well plate to get a final concentration of 2.8 µM at DIV 3-4. However, I was wondering if there are other protocols.
Do you use a higher volume? Do you exchange a part of the medium and add fresh medium together with AraC?
Unfortunately, most protocols only say "addition of AraC" but not the exact procedure.
Thx for your help!
protocol can change depending the study and the question researchers are investigating. if you want to avoid astrocytes presence in your neuronal culture I would say that you should add Ara-C as quick as possible in order to stop their proliferation (for example, 5µM at DIV1, the day after plating). with this protocol, your neuronal culture will be astrocyte-free, therefore neurons will be quite fragile after a long time in culture...therefore if you want to use old neurons for your experiments but it is not an issue to have some astrocytes, your protocol could be more appropriate.
Dear all,
I now use the following protocol:
I take out 100 µL medium from each well (neurons are cultured in 24-well with 500 µL medium) and use this to make a pre-dilution of 14 µM AraC. Then I pipette 100 µL from this solution back in each well. This is how I achieve a final concentration of 2.8 µM in the well (more or less because there might have been some evaporation). I add the AraC at DIV3. At DIV8 I change the medium.
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