Can anyone suggest a protocol for glucose stimulation of rat beta cells for insulin measurement. I can see final glucose concentration as 16,7mM but I dont know why?
Hi Annette, I used to stimulate Min6 cells with 25 mM glucose. Since they are cultured in high glucose medium (that is more or less 25 mM), I cultured them in low glucose for 30 minutes (3 mM) before starting the stimulation. Moreover, since I was measuring Ca2+ oscillations, I used to stimulate Min6 with 25 mM glucose + 15 mM Tetraethil ammonium chloride (TEA) that is a blocker of potassium channels. Thus, I could trigger Ca2+ oscillations and hypothetically insulin secretion.
16.7mM (final concentration) is generally considered the highest range of physiologically relevant (correlates to about 300mg/dL I believe), though i have seen published studies with up to 25mM (closer to 500mg/dL - relevant for mouse/rat levels after glucose administration) as mentioned above. You should definitely culture them in low glucose (2.8mM - 3mM) for a while (I do an hour) before stimulating. Stimulation for 15-30 minutes is probably sufficient to see robust GSIS in these conditions.
MIN6 is a murine cell line and not very responsive to glucose. Therefore, only low passages should be used for secretion experiments (up to about 30).
Seed the cells about 2 days before the experiment. To achieve good secretion, cells should reach about 50% confluence on the day of the experiment. Overconfluent cells don't respond well.
Incubate the cells overnight in low-glucose containing medium (e.g., 3 mM). On the day of the experiment, prepare secretion buffers containing BSA (we use 0.1% BSA). As it was suggested earlier, pre-incubate the cells for 30 min in the incubator (37oC, 5% CO2) with the low-glucose containing secretion buffer (e.g., Krebs buffer). Stimulation for 15-30 minutes or more is sufficient to induce GSIS. 16.7 mM is a good glucose concentration, but you could use also other concentrations. The supernatant is then collected without disturbing the cellular layer, since cells contain high amount of insulin. The cells are lysed and analysed later for the measurement of total insulin content.
In my experience, not every culture is responsive, so it is possible to make a small asay to make sure the control responded before committing to large secretion study.
I hope this helps,
Good luck,
Julia.
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