I'm trying to measure apoptosis levels and reactive oxygen species generation in retinal ganglion cells generated from induced pluripotent stem cells. For both protocols I use Tryple Express to obtain single cells, I filter them through a strainer and I perform all the centrifugation steps at 200g and 4ºC for 5 minutes.
For apoptosis, I use a kit with Annexin V and propidium iodide, and even the kit recommend 5ul of each in 100ul of sample I usually add only 3ul. For ROS, I use DCFH-DA at 10uM for 20 minutes.
I don't know why, I obtain a very high number of annexin V-positive cells (80-90%) and the same for the ROS protocol... I think that maybe I could use a higher dilution of DCFH-DA... but maybe something technical in the protocol is prompting my cells to undergo apoptosis. My thought is that my cells are suffering a lot for some reason during the processing of the sample.
I don't know if someone has experienced the same or if you imagine what could be happening.
Thank you very much in advance.
Hi Marta,
Typical problem with IPSC-derived neurons is depolarized resting membrane potential (~30 mV). At such depolorized resting membrane potential voltage-gated sodium and calcium channels open and can initiate apoptosis or expression of apoptotic genes. I would recommend that you measure the resting membrane potential of your retinal cells, prior to testing ROS and Annexins. Normal resting potential for healthy retinal neurons is about -60 to -70mV; and in your case should be at least -50 mV at which voltage-gated channels are still mostly closed.
best wishes,
Refik
External glutamate in the media and insufficient glutathione/cystine can also cause oxidative stress in neurons. What media are you using for your cultures?
Refik Kanjhan Thank you for your response. At the moment I do not have the possibility of measuring this potential, but I will take it into account.
Robert Adolf Brinzer I am using DMEM-F12 with N2 supplement, B27 supplement without vitamin A and fibroblast basic growth factor. I understand that maybe it could be there some oxidative stress or apoptosis but the thing is that almost all the cells in my experiment are possitive for those markers...
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