Growth and Media Conditions
Confluent cells are split 1:4, 1:6, or 1:8 to 12 well dishes (from 15 cm plates). Media is changed every second day (2 ml per well, RPM1/11 mM glucose, 10% FCS). The day before confluence, the media is always
changed. (2 ml per well (smaller volumes seem to decrease insulin response), RPM1/11 mM glucose, 10% FCS).
At confluence, GSIS is performed. Cells maintain their ability to respond to glucose up to 2 days after confluence if media is changed every day. In contrast, cells not yet confluent will display a weaker response.
Cells are washed gently in 1 ml of SAB w/ 2.5 mM glucose (leaking/damaged cells yield high basal levels and a low fold response)
Cells are preincubated with 1.5 ml of SAB w/ 2.5 mM glucose for 2 hours (preincubation time should not be shorted since this will give rise to a higher basal secretion and subsequently lower fold response)
SAB is replaced by 1.5 ml SAB containing 2.5 mM or 12 mM glucose. Incubation is performed for 2 hours (shorter incubation time, or smaller vol will give lower fold response).
500ul of cell supernatant is transferred to multiplate and centrifuged before RIA. Analysis is performed on 200ul sample (or 100ul + 100ul PBS).
Typical responses are 8-20 fold for clone 13 and 7-10 fold for clone 15
20 islets in 300ul (can scale up, but hard to be accurate with fewer than 20 islets)
Max 200ul sample/tube
For cells - 200ul standards
- 50-100ul sample (1 ml/well in 24 well; 1.5 ml/well in 12 well)
- do not need to normalize volume with PBS
For islets - Usually do not need to dilute samples
- Also don't have much sample to work with. I use 100 ul standard, 100 ul sample (could make additional standard curve if necessary)
1. 1 ml 125I-Insulin, left in tube = total counts (Normal polypropylene tubes)
2. 1 ml 125I-Insulin, dumped out before assay = nonspecific binding (Normal polypropylene tubes)
3-9. Standards A - G
Program 4 = duplicates of standards (2 total, 2 NB, 2 As, etc)
Program 5 = single standard (this usually is fine)
GSIS Assay Protocol
1. Plate cells into desired number of plates with desired number of wells, with appropriate media; begin GSIS assay only once confluent within the wells. (If cells are already confluent before the time the assay will be performed, they should be given new media)
2. To begin the GSIS assay, prepare the 1x SAB mixture, according to amounts shown on the protocol sheet in the lab
a. For 100 ml (sufficient for 1 24-well plate), add to graduated cylinder, in order.
i. 10 ml 10x SAB (found on lab bench)
ii. (in hood!) 2 ml 1 M HEPES (found on lab bench)
iii. 1 ml 0.25 M CaCl2 (found on lab bench)
iv. (In hood!) 0.57 ml 35% BSA (0.2%) (found in fridge door)
v. 0.21 g NaHCO3 (found on dry materials shelves)
vi. H2O to a total volume of 100 ml
c. For other volumes (ex. 150 ml, for 2 24-well plates) simply multiply all volumes by appropriate ratio
3. Divide the SAB mixture into two beakers, in a 1:3 ratio (ex. for 100 ml, 75 ml into one beaker and 25 ml into the other);
4. Calculate amount of glucose (found in fridge door) needed to make the larger-volume SAB mixture 2.5 mM for low glucose, and the smaller-volume SAB mixture 16.7 mM for high glucose (ex. for 150 ml total SAB mixture, this means 100 μl glucose goes into the 100 ml to make the low glucose, and 334 μl glucose goes into the 50 ml to make the high glucose) (So 1 μl glucose/1 ml low, and 6.68 μl glucose/1 ml high)
5. (In hood!) add the appropriate amount of glucose into the beakers. Be sure to label which is which
6. Aspirate media from plates
7. Pipette 0.5 ml low glucose solution into each well (for a 24-well plate. For anything else, I’m not sure how much) (pipette all additions gently onto side of the wells to avoid disturbing the cells)
8. Let stand for 10 minutes.
9. Aspirate off
10. Pipette another 0.5 ml low glucose solution into each well (for 24-well plate)
11. Incubate in tissue culture incubator for 2 hours
12. Aspirate off
13. Pipette 0.5 ml low glucose solution into each well (for 24-well plate)
14. Incubate for one hour
a. During this interval, number/label microcentrifuge tubes and wells so that each well has a corresponding tube
15. Pipette all solution (~1 ml) out from each well into each corresponding microcentrifuge tube.
16. Centrifuge all tubes at 1.5 g for 5 minutes.
17. Prepare a new plate of the same size and with the same labels as the first
18. Pipette 0.4 ml solution from each microcentrifuge tube into each corresponding well of the new plate, avoiding any cells collected in the bottom
19. Seal new plate in Parafilm and store in -20 freezer
20. Pipette 0.5 ml high glucose solution into each well (for 24-well plate)
21. Repeat steps 14 – 19
22. Pipette 100 μl plain RIPA into each well of the original plate (for 24-well plate)
23. Seal original plate in Parafilm and store in -20 freezer
So in the end you have 3 plates if you had one plate originally: one containing insulin in response to low glucose, one containing insulin in response to high glucose, and one containing RIPA with cells.