I have tried to do glucose curves on INS-1 cells using the Mercodia rat kit (https://www.mercodia.com/product/rat-insulin-elisa/) but for some reason the results of the Elisa look inverted. i.e low glucose (3mM) show higher concentrations of insulin than high glucose (18mM) or high glucose + KCl (30mM). This has happened twice. My guess is that dead cells at the low concentrations are contaminating the results ( bursting and releasing insulin).
I culture cells in 24-well plates until 70% confluency for 3-4 days and then do 1h starve in KRBH, followed by treatment with different glucose concentrations for 2h and I take the supernatant.
Has anyone had the same issue or can provide a protocol? Thank you so much
Kindly check these links
Metabolome Response to Glucose in the β-Cell Line INS-1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624472/
Development of a Rapid Insulin Assay
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743966/
...incubated rat INS-1E cells with 0.1, 1 or 5 μg/ml ... rat insulin ELISA Kit from Crystal Chem (Elk Grove Village, IL, USA)
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213650
Chinaza Godswill Awuchi Thanks for your answer. From those links I get that either collecting a smaller amount of supernatant or centrifuging them could help. I will try that. I was also wondering if someone had run into the same issue running this assay. Did you encounter that specific problem while doing the assay yourself?
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