1) 3-5 days prior to experiment, seed 20,000 MIN6 cells per 96 well plate (Corning CellBind).
2) The day of the experiment, prepare fresh KRBH with 0.1% RIA grade BSA.
3) Flick media from plate. Blot briefly on paper towel.
4) Add 150 uL of KRBH + 2 mM glucose to each well (multichannel preferred). Add to the side of the well.
5) Repeat step 3 and 4.
6) Rest cells for 2 hours in the tissue culture incubator.
7) Repeat steps 3-5.
8) Incubate for 1 hour in the tissue culture incubator.
9) Remove 50 uL of supernatant taking care not to disturb the bottom of the well. Spin at 1500xg for 5 min, 4 degrees. Take 25 uL of the supernatant off and freeze at -20.
10) Repeat step 3.
11) Add in KRBH + 20 mM glucose.
12) Repeat steps 8-9.
13) To get total insulin, add 100 uL of lysis buffer.
14) For ELISAs, we use Mercodia Mouse Insulin ELISA kits -- typically, 1:10 dilutions of the supernatants should be in the linear range of the assay. For total insulin, at least a 1:10,000 dilution is required.