The optimal concentration is heavily going to depend on what type cell system you are handling. The 'excess glucose' still has to be below lethal concentrations, in fact at sub-lethal concentrations. Therefore u may want to do an LC50 test.

HI,

I agree with Kumar and the concentration of D-Glucose concentration will vary accordingly based on different cell lines. It's better to conduct a preliminary pilot study to define exact concentration for all future experiments and It makes you job easy for next experimentation.

Hi Elisa,

Like Apurva and Upendarrao - I agree that it will be highly dependant upon the type of cell you use and the glucose concentration that that cell is used to. A lot of cell lines originally were selected in High glucose DMEM or an equivalent - usually 25mM glucose. I would argue that cells selected under these conditions will never be good models for hyperglycaemia because they are selected to survive in supraphysiologically high glucose concentrations that are way beyond what would ever occur in vivo even pathologically.

If you aren't already using a cell line in low glucose then I would suggest you utilise a cell line which grows happily in a low (5mM) glucose medium. Then from there I'd probably do 10mM, 15mM, 20mM, 25mM and 30mM dose curve.

The other thing you might want to consider is exposure to insulin/growth factors also - are you going to be using serum starved, 10% FBS or Donar HS?

Hope this helps.

Thank you all for your help!

I'm using human cells that grow in DMEM low glucose (5mM) and 2% FBS. I started my experiments using 30mM Glucose for the hyperglycaemic model, and my cells after 72h are very nice! I was afraid they would be dead, but instead they don't.

Elisa

How much mL you use at 75cm2 bottle cell culture? My cells doesn't death and anyone oxidative damage was observed!!!

Can we use MTT assay to check the viability of cells at different glucose concentrations?? So that we can optimize the maximum glucose concentration that would mimic the diabetic/hyperglycemia conditions depending on cell line.Can anyone help me with this???

@Muralidharan - MTT may not be good but quick and easy. It's basically tetrazolium salt that gets reduced to formazan compounds by dehydrogenases/reductases in the cytosol and by NADPH in the mitochiondia. If you see a color change then presumably there is something wrong with the cell. And you need not necessarily see cell death with hyperglycemic condition. It's its metabolism that differs. It's the functionality of the cell you should look at. And again as the others have mentioned it depends on the cell type you use. For instance if you're using endothelial cells, it's better to look at their migration and proliferation potential. MTT is not the best for it as some of them report in the literature. I use Brdu assay and it works good for me. Gd luck. gopi

Hello all,

I would like to ask my question as well. I'm doing hyperglycemic condition in cardio myoblast cell line (H9c2). We followed conditions from many published papers. We measured cell viability by MTT method and the results found that D-glucose (at any concentrations) increased cell proliferation, rather than cell death, which was reported in previous publications. So, I'm wondering what we did wrong.

We try to solve the problem by reducing the serum concentration in culture medium (from 10 % FBS to 2.5%), but the cells still happy and grow faster than control (without glucose) group.

Could any  of you suggest us please?

Dear Sarawut,

Have you tried to see if glucose at your tested concentrations change the absorption of MTT without cells?

Good Luck!

When you talk about concentration of 25mM, what is the exact unit?

25mM/L or 25mM/mL

Would be grateful for answer!😉

@ Michał Szymon Krawczyk : To make it more clear, you can prepare 1M stock solution by dissolving 180 grams of glucose in 1 Litre of water. So, if you want to make a working standard of 25 mM, calculate accordingly. Hope this helps you  !!!

Best,

M

Thank U @Muralidharan V😉

I think that there is a paradox for choosing exact D-glucose concentration to induce hyperglycemia! I tried following some published papers to induce hyperglycemia in PC12 cells! The dose was 25 mg/ml. I made it even 50 mg/ml. But the cells look good in this elevated concentration. What the hell is happening? Why I can not reproduce the publish data? Any idea please!

Dear Dr.Sikder,

I afraid that we are facing the similar problem. I did the experiments by incubating cardiac myoblast cell H9c2 in 33 mM D-glucose in DMEM+10% FBS. The cells look good and also induce cell proliferation greater than control DMEM medium. However, cell in hyperglucemic condition seem to more sensitive to stimulant than control cell. In my case, cell in jyperglucemic condition seems to sensitive to injury than control cell.

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!!!