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3D Assay in Matrigel

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 37oC 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

    1. T4-2.

0.7 x 106 cells/1.2 ml matrigel.

    1. T4-2 +AIIBII

0.7 x 106 cells/1.2 ml matrigel

    1. T4-2 +

 

  1. On ice, add 1.2 ml matrigel to tube.
  2. Carefully pipette up and down to distribute cells but not introduce bubbles into the matrigel.
  3. Transfer matrigel and cell mixture to pre-coated 35 mm dish.
  4. Place in 37oC incubator for 15 to 30 minutes and allow matrigel to polymerize.
  5. 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

 

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