You may have already considered the following:

• Make sure your medium does not contain insulin
• Check to see that the apparent transport is not the consequence of a compromised membrane integrity
• A basal level glucose transport should be present, make sure that you are not using too many cells such that the basal transport is causing collectively enough glucose transport into the cells to reach the upper end of your detection limits, assuming that is what you are observing.

I have no idea about where one might get pig tissues in Israel. In the U.S. and many other countries, it is not an issue. You may consider a collaboration with some one in a European country who may be willing to supply you with the tissue.

Best...  

hi, there's always a minimal uptake of glucose in non-treated cells. The major understanding behind the colorimetric method of estimating glucose uptake is, we check for enhanced glucose uptake above the minimal level observed in the non-treated cells. In case of insulin treated, the uptake will be highly enhanced and it is not in case of cells not treated with insulin. Please make sure that the insulin is not spilled while carrying out your experiment. I also totally agree with Dr. Tausif Alam pertaining to the cell count. Please be sure that u seed the required no. of cells equally in all the wells.

Hi

Which method or kit are you using for glucose uptake? 

In my case, washing the cells properly to remove unused 2-DG and placing them on ice during the process helped to reduce high 2-DG values in the controls. 

Thank you all!!

Dear Tausif,

How should I check the membrane integrity?

How many cells do you recommend to use?

We can get pig tissues in Israel :) . I meant, if there is a cell line like 3T3 cells but from pig origin? or is there a simple way to produce adipocytes from pig's tissue?

Dear Snehaa,

Which kit are you using?

When you say "non-treated cells", do you mean cells that were not treated with insulin but were treated with 2DG?

What is the concentration of insulin that you are using?

Dear Madhur,

I'm using Abcam's kit (ab136955). In which step do you place the cells on ice?

Do you use 3T3 adipocytes? If so, how many cells do you seed? Do you differentiate them on the same plate that you are performing the glucose uptake assay?

Hi, Firstly, We din't use kit. 

secondly, yes not treated with insulin but treated with 2-DG (10mM)

And last, Insulin used is 10nM for stimulation and 100nM for positive control

Thanks Snehaa,

Do you do RI assay?

Which kind of cells do you use?

Hello Anat,

1. Any commonly used dye exclusion method would suffice. Examples of such dyes include Eosin, Trypan Blue, and Erythrosine. I prefer Eosin. If the cell membrane is substantially intact, the dye will not enter the cells and this can be confirmed on a microscope (one typically uses these dyes to count live/dead cells). If the cells do take up the dye, they have serious membrane damage. Converse in not always true so if you see that none or the majority of cells are excluding dye, it may not always mean that there isn't damage which is not quite readily detectable.

2. In case of adherent cells, most people perform such test directly in 96-well plates. I have not used Abscam kits but I'm sure they must have a recommended range of cells. Normally, depending on the cells, you may have 10,000 - 50,000 cells/well in a 96-well plate. Ages ago, I have done glucose transport assays in islets (in suspension) using 3H 2-deoxy-glucose. The reason for chilling (without letting the temperature drop below the transition point where the fluidity could be influence) in that case was to control the transport rate to get decent numbers for kinetic measurement.

3. You may have to do some literature searches; I expect that by now someone must have generated pluripotent cells from pigs. Since terminally differentiated cells tend not to grow, an easier way may be use pluripotent cells and differentiate them into adipocytes. Unless, of course someone has cells analogous to 3T3 from pigs.

Best.

T

Hi Anat

I have used this kit for human adipocytes and indeed was bit difficult to standardize. 

As far as I remember, we need to perform the assay in 96 well plate which includes growth and differentiation of cells to mature adipocytes. we seeded about 2000 to 2500 cells per well considering slow growth of these preadipocytes. Place the cells on ice as soon as you wash cells with PBS and about to proceed to NADP degradation. and make sure you wash gently not to lyse or wash off any cells.

But later we moved to radioactive Glucose uptake which is easier and more reliable in our case. Using this kit was bit difficult all together

Hi Anat,

I used 3T3-L1 adipocytes and did not do RI assay

At what temperature do you recommed to incubate the cells with the sample/insulin and at what temp' with Deoxyglucose?

Anat,

Incbation at 37 deg Celsius in CO2 incubator for 30 minutes was done after treatment.

Thanks Snehaa.

Does someone have a recommendation for colorimetric or fluorometric glucose uptake assay kit?

Dear  Madhur,

Hi 

We bought some primary human adipocytes from LONZA.

Also, we isolate the cells from human adipose tissue that were obtained during bariatric surgery. 

All incubation temperatures have to 37 with insulin and deoxyglucose so that cells work at maximum efficiency. 

HI,

Thanks!

Hi all,

Which kind of insulin do you use? And how do you dilute it? With PBS?

We tried several commercial insulin and we got glucose uptake in 3T3 adipocytes only with 600 microM.

Hi Anat Hofshi,

I supose that you have already figured out your problem, but I wish know if you could mesure glucose uptake by a colorimetric method. I tried to do it in muscle cells (L6) but i couldn't. Finally I did it by a radioactive method.

Talking about the insuline, I use a porcine insulin powder from sigma. I dissolve it in acidified milli Q water (pH 2-3) And it works very well. I add insulin to my cells in a final concentration of 100nM.

HI Anat,

I successfully used a colorimetric kit from Cosmo Bio, to measure glucose uptake in muscle:

https://www.cosmobio.com/contents/2_deoxyglucose_uptake_measurement_kit.html

There's also a way to do it without a kit, the method is very well described in the attached paper (though I never tried by myself).

Hi, pls any body provide me some paper which use absorbance or fluorescence method for glucose uptake.

Anand,

check the paper reported in my previous post.

Indirect method of glucose uptake can also be performed. We have been doing it. First estimate initial glucose concentration in media and at end of culture again measure glucose. Difference is glucose uptake.

Does anyone know of a protocol for glucose utilization assay for muscle homogenate? We were able to get the in vitro assay to work with palmitate but we cannot get it to work with glucose. Thanks in advance

(we would like to minimize the use of the in vivo injection method)