QCM Chemotaxis Cell Migration Assay, 24-well (8 µm), colorimetric

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 300 µL of prepared cell suspension from step 3 to each insert. Add 500 µL of serum free media in the presence or absence of chemo-attractant (e.g. 10% fetal bovine serum) to the lower chamber. Cover plate and incubate for 24 hours at 37°C in a CO2 incubator (4-6% CO2)	Measure the Optical Density at 560 nm.

Protocol tips
Add 300 µL of prepared cell suspension from step 3 to each insert. Add 500 µL of serum free media in the presence or absence of chemo-attractant (e.g. 10% fetal bovine serum) to the lower chamber. Cover plate and incubate for 24 hours at 37°C in a CO2 incubator (4-6% CO2)
Downstream tips
Measure the Optical Density at 560 nm.

Publication protocol

Migration ability was analyzed by wound-healing assay and transwell migration assay. FaDu cells (4 × 105) and Detroit 562 cells (5 × 105) were seeded into 24 well plates. When cells reached a 90–100% confluent monolayer, a scratch was made by a 200 μL pipette tip. Cell debris was removed by phosphate-buffered saline washing after scratching and then cells were incubated in MEM medium with 2% FBS. Images were taken at 0, 12, 18, and 24 h after SPARC treatment. Transwell migration assay was performed in the QCM™ 24-well Cell Migration Assay and Invasion System uncoated 8 m pore size polycarbonate membranes (Millipore) according to the manufacturer’s instructions. Briefly, 8 × 104 FaDu or 1 × 105 Detroit 562 were seeded into 24 wells and inserted into 300 μL serum-free medium while 500 μL medium with 10% FBS was in the lower chamber. After 24 h, the bottom surface of the membrane was fixed in 10% formaldehyde solution, followed by 0.4 g/L crystal violet staining for 2 h. Cells on the upper surface were removed by a cotton swab after membrane washing. The bottom of the membrane was then visualized using the Olympus inverted microscope at 100× magnification. Four random fields of view were counted and the relative fold of migration in each group was compared to the 0 ng/mL SPARC treated group.

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