I have been trying to do a glucose uptake experiment using fluorescent dye to measure if my protein of interest will have impact on glucose uptake. From experience, what is the best cell line for this? Do you add insulin? Do you starve your cells?
Hi Dr
different researchers us different type of cells according to their needs such as
3T3L1 cells, C2C12muscle cells, L6 cells, PC12 cells
Thanks & Regards
Hi Dr
different researchers us different type of cells according to their needs such as
3T3L1 cells, C2C12muscle cells, L6 cells, PC12 cells
Thanks & Regards
If you're looking at insulin-dependent glucose uptake then a metabolic cell line such as C2C12 or L6 is fine. We use these and also differentiated 3T3-L1 cells. We use 2-NBDG and glucose oxidase assay of culture supernatant.
Depends! Target tissues are adipose tissue and muscle. L6, C2C12, 3T3L1 are great You can also use an alternative cell model (circulating monocytes, not cell line) that produces excellent data. Please see
http://www.ncbi.nlm.nih.gov/pubmed/15688355
http://www.ncbi.nlm.nih.gov/pubmed/20643758
http://www.ncbi.nlm.nih.gov/pubmed/18299470
http://www.ncbi.nlm.nih.gov/pubmed/17373964
Best of luck
Thanks for the answers. What if you are interested in the insulin indepindant glucose uptake James?whould you not add insulin with the 2-NBDG
Hi Mohamed. 2-NBDG transport isn't hugely responsive to insulin, at least not as much as glucose is. That is why we measure both 2-NBDG (or 6-NBDG) uptake and the amount of glucose lost from the media :)
But what does the media give you in this case? Do you just take the media and mesure it in the plate reader? how do you recomend to proceed in this case?
If your sell culture has 4.5g/L of glucose in it at the start if an experiment you can measure long-term glucose uptake by measuring how much the glucose level has dropped. It won't be sensitive for short-term measurements though. Are you going to also block GLUT channels to provide a mechanism?
The gold standard used to be primary adipocytes obtained from epidydimal fat. This was replaced by differentiated 3t3-L1 cells and other assorted lines. Back in the days we used radioactive 2-deoxyglucose, which behaves just like glucose for uptake. One thing that worries me about your assay is that James stated that the uptake of the dye is not insulin sensitive. This clearly suggests that the uptake mechanism is not the same as the mechanism of uptake of glucose. In other words, it is probably not getting in via GLUT4. If this is the case, your assay may not be the most convenient. As to measuring the depletion of glucose in the medium, I am not sure how sensitive this assay is.
Guillermo is absolutely right, the most sensitive assay would be isotope based but radioactive work seems to be very much out of fashion today. 2-NBDG will be transport in response to insulin, we do see an increase in cellular fluorescence in L6 cells but as the attached graph shows not as much as when stimulated with substance X. Measuring glucose in the culture supernatant is accurate but only for long term incubations (we've measured significant differences in astrocyte populations. Not much good for kinetic or dynamic observations but really easy/cheap for chronic observations.
Hi Dr. Abu-farha,
We use [3H]2-deoxyglucose to measure glucose uptake in our cells. We use glucose free media on the cells (there's various media types that you can purchase that are glucose free). We change the media to the glucose-free one for several hours before adding the labeled glucose. Here's a link to a paper our lab published that describes the assay in further detail. http://cancerres.aacrjournals.org/content/65/2/613.long
Good luck!
Adipose cell and muscle cell are responsible for glucose uptake.
3T3-L1 adipocyte is the golden cell line to glucose uptake assay. L6 cell is ok.
Primary adipocytes are much more responsive than 3T3-L1, but they are harder to transfect.
Hello Dr Mohamed.
Most of the time researchers used 3T3 Cells, L6 cells, the EAhy 926 endothelial cells and C2C12 muscle cells. The 2 last cells are the one i have been using.
HI Dr. Mohamed
I think most of researcher like 3T3-L1 or C2C12. Dependent on your experiment design and what kind of question you want to ask? These two cells both can response to insulin.
Hi Dr. Mohamed,
To understand the complete glucose homeostasis, one should check glucose uptake in both L6 (glycogen formation) and 3T3-L1 adipocyte (lipid formation) cell lines.
[3H]2-deoxyglucose is an excellent marker for measure glucose uptake in proximal tubular epithelial cell preparations whether in isolated vesicles form the apical membrane which exhibit a great inside transport of glucose either sodium-dependent or independent transport. LLCPK1 cell line is also a good model. Starving is not necessary, withdrawal of serum from the culture media 24h before your experiments assures you better results. These transporters are independent from insulin.
For sure I would use 3T3-L1 adipocytes. You can see up to 4-5-fold increase in insulin-stimulated glucose uptake in these cells. Much more than in L6 muscle cells or in PC12 cells.
Has anyone done this assay with neural derived cell lines with an easy budget friendly method or kit? how protocol changes when not usin muscle and lipo cells?
I have not doen this assay, but its best to start with an insulin dependent cell line to prove you have active glucose transport. This can be your positive control and you can standardize optimum conditions to visualize your fluorescent probe. Then, you can work on your protein of interest.
Primary adipocytes are the gold standard for the assay. Do not used C2C12, they have to much passive transport, L6 and 3T3L1 can work depending on how you keep the cells. good luck.
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