Hello!
I need to develop a Diabetic Retinopathy in vitro model with RPE cell line.
I will use 25 or 30mM HG (D-glucose) and 5mM for Normal Conditions (NG).
However I have a lot of doubts:
My DMEM-F12 Gibco formulation have 3.125mg/l
Hence:
1) to obtain 5mM and 30mM I have to consider the basal levels of glucose?
2) I will do a MTT to have the IC50, do you think that it is much better without FBS (0%)? For how many time? 24hs?
Thank you!!!
Yes I can..but the thing is that I want to have a good DR in vitro model.
use several concentrations of glucose and do not rely on MTT assay alone for determining IC50 of glucose. Instead, use a combined CV/MTT/NR assay as described elsewhere. You may additionally use Alamar Blue assay; although the principle is the same, the dye used in this assay is non-toxic unlike MTT. You may also use BrdU assay. The intention is to be able to finely understand and model the toxicity of a largely non-toxic agent i.e. glucose.
You should include or exclude FBS depending on the conditions and protocols under which you plan to use the in vitro model. You must be planning to test certain drugs or agents in the in vitro model. If testing those agents requires addition of FBS to RPE cells, then you should include FBS at this stage as well.
You may refer to the time-frame indicated in the specific protocol for CV/MTT/NR assay; 24 hours should be sufficient -- the basic idea is to allow the cells enough time to go through the log phase.
Bhaskar
Thank you for your response...I used mtt neutral red ans trypan in othee modwls during my PhD Thesis. However now I want to initiate a new research line and I hope to find someone that use Diabetic Retinopathy in vitro model that could give me infomation. The control 5mM is the most doubt that I have because GIBCO formulation have glucose..why I have to add nore glucose?
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