- 90% confluent monolayer of cells in a 24 well plate.
- 10 microlitre micropipette tips.
- Culture Media.
- Inverted microscope.
- Cell Culture media.
- 5% dexpanthenol containing media.
- Hight FBS containing media.
Prior to the assay:
- Cells should be cultured to confluence or near (>90%) confluence in 24 well dish.
On the day of the assay:
- Prepare 10 ml of 5% dexpanthenol containing media along with 10ml of high FBS containing media.
- Using a sterile 10 μl pipet tip, scratch three separate wounds through the cells moving perpendicular to the line drawn in the step above. See the figure for arraignment of the scratches.
- Rinse the cells (very gently as sheets of the cells may lift off if you are not careful) with PBS and replace 8 wells with 1 ml of 5% dexpanthenol containing media, 8 wells with High FBS containing media and 8 well with normal cell culture media in the order shown below.
- Take a picture using phase contrast and 10X at 0 hours. Carefully label the pictures.
- Take pictures at 2, 4 and 6 hours.
Difference Encountered in a Real Laboratory
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
1. Before starting the experiment sterile the laminar air flow chamber using spirit.
2. Always disinfect your work area when you are finished.