I am trying to do GSIS using MIN6 cells and I am measuring secretion via ELISA; surprisingly I am obtaining higher insulin secretion when I am stimulating the cells with low glucose concentration (3.3mM) than with high glucose concentration (16.7mM).
can anyone please help ?
Dear Nancy,
I did few static incubation on Min6 cells but I did a lot on rat, pig, mouse and human islets and isolated primary beta cells. I don't exactly know what is your protocol but one thing that you can do (if you are not already doing) is to do a sequential static incubation. Normaly, you are cultivating your cells in low glucose to avoid depletion of the insulin stocks. So to start you incubation, wash your cells in PBS for 5 minutes then put your 3.3mM medium for 30 to 45 mins. Collect your medium and add the one at 16.7mM for the same period of time. Collect your medium and finish with a last incubation at 3.3mM for the same time and collect. Once you did the ELISA (Mercodia is the best one for me :-)), calculate your stimulation index by: qtity of insulin @ 16.7mM/((qtity of insulin @3.3mM n°1+qutity of insulin @3.3mM n°2)/2). It will also help you to evaluate how your cells are behaving. For your problem of insulin quantities, check also if you don't have detached cells @3.3mM that could explain the higher insulin. If those cells are degraded, they will release intracellular insulin. a last test that you can do to be sure that your cells are responsive in doing, at the same time but in separated wells, a condition @16.7mM and another one @ 16.7mM+forskolin. Normally, cells should release more insulin with forskolin.
I hope that it will help you to find the answer to your problem.
Regards,
Thomas
Hey Timothy,
I appreciate your reply. I will try to wash the cells efficiently hoping for better results.
Dear Thomas
I deeply appreciate the detailistic clarification that you provided. i will proceed with my experiment accordingly andI will keep you updated with the results.
Regards
nancy
In an ideal situation, one would perifuse insulin producing cells but that makes for a somewhat complicated experimental setup. In static incubations, one must always go back to low or normal glucose at the end to demonstrate that there is no (or insignificant) cell death or detachment (always gently spin the collected medium to remove detached cells), giving misleading numbers.
One of the possible reason can be the degradation of cell. Are your cells looking healthy in the entire process? I do agree with Timothy- washing with PBS is a good addition with lower speed. Are you considering 5.5mM glucose concentration as the basal? In that case plz check the insulin secretion at 5.5mM Glucose before and after the GSIS. Any big variation will indicate the possible change in MIn6 number.
Also depending on your ELISA kit sensitivity you can do dilution.
Hi Nancy,
I had the same problem before. I didn't spin down the supernatants I collected before insulin assay. So if you don't spin down the supernatants perform this spin down at 2000 rpm for 4 minutes. I did so and it made a big difference. Although you do washing, few cells still remain in the supernatant and can cause higher insulin readings at low glucose treatment.
I am about to do the GSIS for MIN6 cells. Does anyone have an idea about the detailed protocol used by our lab's neighbors for GSIS for MIN6 cells (see attachment or below)? Any suggestions, comments or critics are welcomed.
1. Wash two times with PBS.
2. Add DMEM media (serum free and no glucose) for 2 hours.
3. Add basal DMEM media contain 1 mM glucose for 30 min.
4. Collect these medium, save at -80°C. This will be the baseline insulin concentration.
5. Add higher concentration of glucose medium to the cells for 30 min.
We added four kinds of concentrations of glucose medium as follows:
12-well plate:
Glucose Well
3.3 mM 1A, 1B, 1C
5.5 mM 2A, 2B, 2C
11 mM 3A, 3B, 3C
25 mM 4A, 4B, 4C
6. 1 hr later, the medium are collected. Keep at -80°C.
7. Wash cells two times with PBS.
8. Add 100 ul H2O and 63 ul lysis buffer (0.18 M HCl in 96% ethanol) to the wells, collect the cells and sonicate, keep for overnight at 4°C.
9. Spin the cell lystate for 5 min at 10000 rpm, keep the supernatant at -80°C.
10. Insulin is measured by ELISA.
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