7- 3D Assay in Matrigel : S1, T4-2, T4-2R
(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 37oC 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
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 37oC 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.