We use fibroblast-conditioned media for all our migration/invasion assays, as we are fortunate to share a lab with a group that routinely cultures fibroblasts. Alternatively, you could use a serum gradient, e.g. seeding your cells in serum-free or low-serum conditions in your boyden chamber, and using full-serum medium in the bottom well. I have done this with both mammary and colorectal adenocarcinoma cells, and my guess is that it should work on PC9 cells too as they rely on EGFR signaling (you could also use recombinant EGF as a chemoattractant, but that's way more expensive).
Hello
For migration assays.
cells were preincubated 3 hours with
10 ng/mL EGF, 1 mmol/L PHA665752, or 1 mmol/L PF-
04217903, then plated at 5 104 cells per well in 24-well plates
with 8-mm filter inserts . Conditions were toward 10% FBS. after that , Cells were stained and cell number was averaged from three fields of view per chamber.
For Invasion assays
were conducted similarly but with 1.4 105 cells per well in
Matrigel invasion chambers .
For growth assays
cells were plated at 1 104 cells per well in a 12-well
dish and counted daily for 5 days.
Thank you Saleh Alkarim, information you gave is alot for me. I have some more concern on the cell number. I used 2.5 10^4 cells per insert, on the last day of assay, the cell density was almost 100% or say overgrowth. So, I reduced cell number to 1.5 10^4 per insert. Does the difference in cell number make any changes in the effective outcome of result or the number of migrated cells changes accordingly with the cell number used??
PC9 cells can self-generate guiding gradients in their normal culture media and display directional movement in various conditions, without the need for a chemoattractant. For example, individual PC9 cells can move along the shortest path through microscale mazes, moving away from larger groups of cells towards unoccupied spaces. Other conditions that impose restrictions to diffusion may help you with your assay.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3440622/figure/F1/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3440622
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