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Invasion Assay

Invasion Assay

 

Materials:

 

Falcon 24 well tissue culture plate

Falcon Cell Culture Insert: 8μm pore size, 24 well format

                                           Cat# 3097

Matrigel (Diluted in DMEM/F12)

5% Glutaraldehyde in 1XPBS

0.5% Toluidine Blue in 2% Na2CO3

 

 Preparation of Boyden Chamber:

 

  1. Using sterile forceps remove cell wall inserts from their packaging and pit them into wells of a 24 wells plate.
  2. Wash inserts 2 times with 300μl DMEM/F12

(Be careful not to stab at the membrane)

  1. Add 20μl of 1:6 diluted Matrigel (2-3 mg/ml protein) to the center of each cell well inserts. Gently spread the Matrigel across the entire surface of the membrane.
  2. Place coated inserts in incubator to allow the Matrigel to solidify for 20-30 min.

Preparation of Cells for Assay:

 

  1. Treat and grow cells according to experimental condition. It may be desirable to synchronize cells to the same growth state.
  2. Trypsinizing  and count of cells
  3. Remove trypsin and resuspend with fresh medium.
  4. Plate 1X105 cells in 200 μl of defined medium into upper chamber.
  5. Add 300 μl of medium lower chamber.
  6. Culture for approximately 20 hours.

The time varies for different cell lines and may be up to 48 hours for less invasive cells.

 

Fixation and Staining:

 

  1. After culture, aspirate medium from lower chamber
  2. Add 0.5 ml of 5% Glutaraldehyde in 1XPBS, incubate for 10 min. @ R.T
  3. Aspirate Glutaraldehyde solution, wash each well 3 times with D.W
  4. Add 0.5 ml  of 0.5 % Toluidine Blue staining solution into the lower chamber, incubate for 10-20 min @ R.T
  5. Aspirate solution from both upper and lower chamber, wash lower chamber 3 times with D.W
  6. Carefully wipe the inner surface of the upper chamber using a cotton swab. (Be careful not to press too firmly, the membrane may be popped out.) Invaded cells will be in the center of bottom of the membrane.
  7. Count the number of cells with q 20X objective of microscope. Count at least 3 fields per membrane.

 

Reference:

 

Andre Lochter, Anavella Srebrow, Carolyn J. Sympson, Nathan Terracio, Zena Werb, and Mina J. Bissell (1997)J. Biol. Chem., 272, 5007-5015