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College of Life Sciences Nutrition, Dietetics, and Food Science
Tessem Laboratory - Diabetes Research Group

Islet Dispersal

  1. Wash islets in PBS
    1. Pick islets into eppendorf with 1ml PBS and spin at 1000rpm for 2 min at room temp. Aspirate supernatant.
  2. Trypsinize
    1. Add 200ul o.25% trypsin/EDTA and place in 37C waterbath for 5 min
    2. Spin tubes (1000rpm, 2 min, room temp) and DECANT supernatant.
    3. Warning: If you use a pipette tip, you will lose the cell pellet.
  3. Wash off trypsin
    1. Add 750ul sterile PBS and spin 3 min.
    2. DECANT supernatant
  4. Plate cells
    1. Resuspend cells in complete media
      1. Use # wells per tube multiplied be 50ul (i.e. resuspend in 200ul for 4 wells)
    2. GENTLY triturate until pellet is broken up
      1. Warning: Be gentle, otherwise the cells will look sick the next day.
    3. Put drops of 50ul into 24 well plate OR 12 well plate with 12mm glass coverslips for staining.
    4. Incubate 37C
  5. Culture
    1. After 1-2 hours, add 1ml complete media gently to the side of each well.