I'm trying to perform whole-cell patch-clamp on primary astroglia cultures and I've noticed a very narrow time window to get good seels (aprox. 3-4 days after isolation). One day I have nice cells and obtaining gigaseal is easy, and next day it is very hard to patch-clamp cells. Do you make the same observations? If not, what can be the cause? Different room temperature?
Dear Joanna, I think that there are several parameter affeting the eficiency of technique (temperature, osmolarity, culture medium…). Depends also of the type of culture that you have. In my experience, using neurons or astrocites cultures, change the culture medium is a good tip to extend the days of that window that you mentioned. In the case of neurons, we wait 7 days after seeded, then we record since 7 until 14-16 days. In the case of astrocytes we wait 7 days after seeded, and then we record since 7 until 12 days. Moreover, we change the culture medium each 3 days, with new medium. Also we observe that the density is crucial, in general we seeded 5-10X10^4 cells/well.
Difficulty in getting giga seals from cultured cells suggests that the cells you are trying to patch probably are not healthy. You may need to play around with what kind of media to use.
Dear Joanna
Are you culturing the neurons, in that case, I would guess that you have trouble with the culture process. You should be able to keep them for weeks.
If it is survival, primary culture, we need to know the age and the type of model you are using.
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