Hello researchers,
I am trying to check the effect of a chemokine on lung cancer cell (A549) metabolism. Last time I tried to induce the cells with different concentration of the chemokine (inducer) at 0,10,20,50,100 and 200ng for 24,48 and 72 hours and check the absorbance of lactic acid (LD) using the colorimetric kit. The values in triplicate at each concentration and time point vary from each other making it difficult to get any idea of what could be the possible optimum dose and/or time point.
My question is do the cells need to be starved before performing this assay? and is it reliable to check LD to describe the glucose uptake? Anyone could suggest me any change in my experimental design or can recommend me a good protocol?
From what I understand, you want to measure the impact of a certain chemokine on glucose uptake. Lactate is the end product of glycolysis. Therefore, by measuring it, you are measuring glucose uptake indirectly. A better approach would be to use a glucose uptake kit, if you are specifically interested in that aspect (links below). From my experience and from what I see, you do not have to starve the cells.
I would expect the values to differ at different time points and different concentrations. If you have issues getting consistent values for triplicates, this is what I would do.
1. Follow the kit protocol to a T.
2. Add a positive control (bacteria or something that induces glucose uptake and glycolysis) so you know your kit works and that your cells are metabolizing glucose.
3. Are you sure your chemokine induces glucose uptake? Is there a reason why you picked the 10-200 ng range? If not, check the literature and see if you should be using a different range.
4. Make sure you have similar cell numbers in your triplicate wells.
5. Pick a dose and time point where your values are in the linear range (preferably on the higher end) of the assay. Values nearing upper/lower asymptote can give you high variability.
6. If still having issues, use a different kit. I have had good experience with Cayman Chemicals.
https://www.promega.ca/resources/pubhub/comparison-of-glucose-uptake-assay-methods/
https://www.caymanchem.com/pdfs/600470.pdf
https://www.caymanchem.com/pdfs/600450.pdf
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