We are trying to test the effect of insulin on cancer organoids. I am planning to try concentrations between 0 nM to 200 nM but I am open for suggestions. We are looking for acute responses, mostly changes in gene expression mediated by insulin. But I am not sure about what should be the ideal time points that I should pick for the experiments - few minutes/hours of insulin treatment prior to harvesting the organoids? How much time does the insulin take to change the gene expression profile of a cell? I would be very glad if anyone has any experience/suggestions. Thank you!
Frequently, in experiments on insulin action in metabolic research, a concentration of 10^-7M is used.
Defining time points is difficult. Which signal is your readout? As your organoids probably are rather precious, performing time course experiments with appropriate cell lines in order to get an idea (and reference data which may be good for discussion) might be a good starting point. Check the literature, what has been published already for individual signals.
Be aware, though, that stimulation experiments starting at starving conditions may not represent the actual situation present in chronic hyperinsulinemia.
Thanks Wolfgang Schechinger .We want to look at general changes in the gene expression or protein profile (RNA Seq or proteomic studies) upon insulin addition. So wanted to know what time point would be ideal to capture these changes?
We might not have a starvation condition but low insulin/glucose in media that matches with physiological levels.
good question, i used 100nM of insulin for only 20 minutes to calculate glucose uptake in skeletal myotubes; the protocol was as below;
Glucose uptake assay
Glucose uptake was measured by the incorporation of tritium (3H) labelled 2-
deoxyglucose (2-DG) into myotubes in culture in the presence or absence of 100
nM insulin. In each experiment, 10 μM Cytochalasin B was added to one of the
wells to measure non-specific deoxyglucose uptake as it causes inhibition of
glucose transport through glucose transporters. 2-DG uptake was measured by
liquid scintillation counting to quantify radioactivity. The radiolabelled 2-
deoxyglucose is not metabolised nor broken down upon entry into the cell. The
assay was performed on 7 day differentiated myotubes that were cultured in either
6well plates or 35mm dishes.
The differentiation media was changed to reduced serum media before the
beginning of the assay. Cells were incubated with or without stimulus that could be
treatment or EPS for a given time at 37°C. After treatment, cells were washed
twice with warm Kreb's buffer, and then cells were incubated in Krebs' buffer (see
Table 2-4) with or without 100nM insulin or cytochalasin B (10μM) for 20 minutes
in the incubator at 37°C and 5% CO2. Glucose transport was initiated by the
addition of hot glucose 50μl of 2-[3H]-deoxyglucose (Final concentration 50 μmol/l
and 1 μCi/ml) to each well for 10 minutes.
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