What are the solvents used to dissolve the standard (Diazoxide, Glibenclamide, Sulphanylurea) drugs for insulin assay of Pancreatic beta cells of rat growing in RPMI media?
I am performing a standard insulin assay using the Mercodia insulin kit. The cells are being grown in 96 well plates at 50k cells per well.
Please also let me know the concentration of these drugs in the standard assay and cell number used per plate.
If possible, please also send me a few good but simplified papers on insulin assays.
Thanks !
DMSO works!
Tolbutamide at 100 uM works with INS1E cells
Endocrinology. 2004 Feb;145(2):667-78. Epub 2003 Oct 30.
If you do not want to use DMSO, you can dissolve diazoxide or tolbutamide as a 50mM stock solution in NaOH 0.1M, then add at 50-250 µM final concentration and correct pH with the same volume of HCl 0.1N.
Like others suggested DMSO works good. 75 uM also works successfully. Good Luck
I did my insulin assay by keeping in mind above mentioned suggestions. But, I got result s only for the standards (calibrator) curve. For samples (Tolbutamide, Diazoxide, Curcumin), all showed approx same result, around the zero calibrator. In, actual no results for the samples.
I used 50k Rat beta cells per well in 96 well plates. Incubated the well proliferated (72 hrs precultured cells) cells with samples in plane RPMI media for 2 hrs and then used the supernatant for insulin assay using Mercodia kit. Even on concentrating the supernatant through lyophilization, no results were obtained for the sample.
Please let me know, where I might have mistaken. What are the necessary precautions I should i take! My cells are growing fine with no contamination.
Thanks!
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