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.)

 

 

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

1.0 x 10⁶ cells/1.2 ml matrigel

2. T4-2.

0.7 x 10⁶ cells/1.2 ml matrigel.

3. T4-2 +AIIBII

0.7 x 10⁶ cells/1.2 ml matrigel

4. T4-2 +

 

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

 

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