Hi ,I used your protocol to make retina explant everything went...

Thank you, I am starting new explant tomorrow, hopefully work this time:)

Hi Maryam, sometimes there is some contamination that can be visible around the explants. It looks like a white ring. The explants still grow and this contamination doesn't take over but it is not optimal to grow explants in these conditions. For the medium, no need to change the composition but make sure you change it twice a week. The evaporation of the medium is quit substential and it depend on the kind of incubator you have. We have an incubator the monitor the humidity to 90% and we calculated that 30 microliters of medium evaporate every day. The protocol says that 80% of the medium should be change but you can change the totality and it won't affect your culture. Hope it helps! Good luck, Christine

Thanks Christine, I will make sure to follow these steps next time. What are the reasons that my culture might be unhealthy? Do I have to switch the media to different one?

Thanks

Hi Maryam,
This can happen. Make sure you are using room temperature PFA (very important). You should first remove the medium from under the filter and replace it with PFA. Fix for 5 minutes and than add PFA on top of the explants. If your explants still detach easily it is often a sign that they are not very healthy.