(Written for 35 mm dishes ? if smaller dishes are used, volumes of matrigel must be adjusted to correct for the tendency of matrigel to adhere to the sides of the dish.)

Pre-coat 35 mm dish with 200 to 300 microliters of matrigel. Place dish in 37o C incubator for 10 to 15 minutes and allow matrigel to polymerize. during polymerization of the matrigel, trypinsize cells. Pellet 0.6 to 1.2 x 106 cells, aspirate media, flick tube to break up pellet.? S1 cells

1.0 x 106 cells/1.2 ml matrigel

T4-2.

0.7 x 106 cells/1.2 ml matrigel.

T4-2 +AIIBII

0.7 x 106 cells/1.2 ml matrigel

T4-2 +

On ice, add 1.2 ml matrigel to tube. Carefully pipette up and down to distribute cells but not introduce bubbles into the matrigel. Transfer matrigel and cell mixture to pre-coated 35 mm dish. Place in 37o C incubator for 15 to 30 minutes and allow matrigel to polymerize. Once gel is formed, add 1.5 to 2.0 ml media to dish. For S1: H14+EGF medium For T4-2: H14 medium To revert T4-2 with tyrophorstin: H14 medium + tyrophorstin 80nM

We grow our cells for 10 days to allow spheroids to form; changing media every 2 to 3 days.