1)      Using sterile/flamed forceps (or sterile stripettes as chopsticks) place the insert (also called upper chamber) into a regular 24-well plate.  Add 100µl of serum-free media (for SUM1315: F12 media only) into the insert and place back into the incubator for > 1hr.

2)      Trypsinze cells and resuspend in serum free media to count.  Establish cell suspension so that 5×104 cells are in 150µl media.

3)      Add 750µl of fully supplemented media (F12+5%FBS+I/E) into wells (also called lower chamber) not containing the inserts of a 24-well plate.

4)      Using sterile forceps (or sterile stripettes as chopsticks), transfer the insert chambers to the wells containing the media. Try to avoid getting air bubbles trapped below the surface of the insert.

5)      Immediately add the cell suspension of 5×104 cells/ 150µl media to the insert well containing 100µl of media.

6)      Incubate for 24-48hours.

7)      After incubation, carefully aspirate the media from the top chamber and transfer the inserts from the well into another well containing PBS to wash.

8)      After washing, scrape the inside of the upper chamber with a cotton swab to remove the cells from the inside of the well.

9)      Stain cells on the underside of the insert by placing the insert into Magic Stain solution for 30 sec.

10)  Wash with water and the air dry.  Count the number of purple cells/field under a microscope.

Crystal Violet (Magic Solution)

For 500ml (dH20 + 20% ethanol)

15g Crystal violet

4.45g ammonium oxalate (oxalic acid, diammonium salt)

Filter before use