Materials:

• Vial of Monocytes (cat. no. 1008 or 1009)
• AIM V® medium
• formyl-methionine-leucine-phenylalanine (fMLF) or other chemoattractant. fMLF is dissolved in DMSO to make a 1 mM stock solution
• Transwell® inserts, 3 um pore size (Corning catalog#3415)
• XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
• HBSS (Hank's Balanced Salt Solution)
• 24 well plates
• Microplate spectrophotometer
• Sterile forceps

1. Prepare chemoattractant in AIM V® medium and add to wells of 24 well plates at 0.6 ml per well. We have found 10 nM of fMLF is optimal.
2. Place Transwells® into wells with medium and incubate overnight at 37°C in 5% CO₂. Also incubate an aliquot of AIM V® for resuspension of the cells.
3. On the second day, check the Transwells® to see that no medium has entered the upper chamber. This would eliminate the chemoattractant gradient necessary to stimulate migration.
4. Thaw a vial of monocytes and adjust the cell concentration to 10⁷ cells per mL in the prewarmed medium.
5. Prepare dilutions of the cells to create a standard curve. Cell concentrations between 100,000 and 10⁶ per mL are most useful.
6. Add 100 microliters of the cells to each Transwell® (equal to 10⁶ cells per Transwell®) and 0.6 mL of cells to each well for the standard curve. Each condition should be run at least in duplicate.
7. Incubate at 37°C in 5% CO2 for 4 hours.
8. Just before the end of the 4 hour incubation, prepare a solution of XTT by dissolving 9 mg of XTT in 5 mL of water and adding 5 mL of HBSS.
9. Remove Transwells® and add 0.3 mL of XTT to each well.
10. Incubate for 18-20 hours at 37°C in 5% CO₂.
11. Read absorbance at 490 nM with a reference reading at 630 nm.
12. Plot absorbance as a function of cell concentration and use the standard curve to determine the number of cells that have migrated.

AIM V® is a registered trademark of Invitrogen, Inc. Transwell® is a trademark of Corning Life Sciences.