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Post date: Jul 9, 2012 4:44:09 AM
3.1 Aims
- Measure invasion of cells through a Matrigel matrix
3.2 Materials
- Coated invasion chambers (see associated protocol)
- 24-well plate
- Chemoattractant (10% FBS with media)
- Starving media (0.5% FBS with media)
- Negative control cells (U87 MGMT)
- Positive control cells (U87 EV)
- Staining kit (diff-quick) or Crystal violet
3.3 Methods
3.3.1 Cell Preparation
- Serum-starve cells for 24 h
- Prepare cell suspensions as follows
o Aspirate media, wash with PBS and add 2 ml of trypsin
o Add 3 ml of serum-starved-media and transfer to 15ml tubes and centrifuge
o Re-suspend in 1 ml serum-starved-media and count the cells
o Bring the concentration to 2x105 cells/ml for each cell line
- Add chemoattractant to the wells (0.750 ml)
- Use sterile forceps to transfer the chambers and control inserts to the wells containing the chemoattractant.
Be sure that no air bubbles are trapped beneath the membranes.
This can be avoided by tipping the insert or chamber at a slight angle as it is lowered into the liquid.
- Add 0.5 ml of cell suspension (1x105 cells)
- Incubate for 18 hours
- After incubation, aspirate media and remove non-invading cells from the upper surface of the membrane by “scrubbing”.
Scrubbing must be accomplished quickly to avoid drying of cells adhering to the bottom surface of the membrane.
- Insert a cotton swab into the insert and apply gentle but firm pressure while moving the tip over the membrane surface.
- Repeat the scrubbing with a second swab moistened with medium.
3.3.2 Staining
- Add each Diff-Quik solution to three rows of a BD Falcon™ TC Companion Plate.
- Add distilled water to two beakers.
- Sequentially transfer the inserts through each stain solution and the two beakers of water.
o Allow approximately 7 minutes in each solution.
- Allow the inserts to air dry.
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