Falcon 24 well tissue culture plate
Falcon Cell Culture Insert: 8μm pore size, 24 well format
Matrigel (Diluted in DMEM/F12)
5% Glutaraldehyde in 1XPBS
0.5% Toluidine Blue in 2% Na2CO3
Preparation of Boyden Chamber:
- Using sterile forceps remove cell wall inserts from their packaging and pit them into wells of a 24 wells plate.
- Wash inserts 2 times with 300μl DMEM/F12
(Be careful not to stab at the membrane)
- 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.
- Place coated inserts in incubator to allow the Matrigel to solidify for 20-30 min.
Preparation of Cells for Assay:
- Treat and grow cells according to experimental condition. It may be desirable to synchronize cells to the same growth state.
- Trypsinizing and count of cells
- Remove trypsin and resuspend with fresh medium.
- Plate 1X105 cells in 200 μl of defined medium into upper chamber.
- Add 300 μl of medium lower chamber.
- 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:
- After culture, aspirate medium from lower chamber
- Add 0.5 ml of 5% Glutaraldehyde in 1XPBS, incubate for 10 min. @ R.T
- Aspirate Glutaraldehyde solution, wash each well 3 times with D.W
- Add 0.5 ml of 0.5 % Toluidine Blue staining solution into the lower chamber, incubate for 10-20 min @ R.T
- Aspirate solution from both upper and lower chamber, wash lower chamber 3 times with D.W
- 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.
- Count the number of cells with q 20X objective of microscope. Count at least 3 fields per membrane.
Andre Lochter, Anavella Srebrow, Carolyn J. Sympson, Nathan Terracio, Zena Werb, and Mina J. Bissell (1997)J. Biol. Chem., 272, 5007-5015