After 3 hours of incubation of cancer cells (in 150ul media 1%BSA) on top of the 8um pore size insert with known concentration of chemo-attractant at the bottom(1ml of media+chemoattractant 1%BSA), I see that the cells migrated more at the edges and less at the center. Since I need to count the cells at 3-4 different locations to quantify cell migration, this is a problem.
Can anyone suggest me how to solve this?
Thanks
Hi Rahul, I had the same problems.
The reason was either the meniscus of the liquid in the upper chamber was too close to the surface and impeded uniform cell distribution. This can be solved by adding more media to the upper chamber.
Or the chambers were not agitated enough to allow for more even cell distribution before they attach.
Hi Rahul,
I always have more cells migrating in the edges but I can count the cells juts in the middle of the chamber using High Content Analysis with the meta-express software. With this methodology you can select the area and count the cells depending of their size and intensity of the color (if you are using crystal violet) or fluorescence.(DAPI).
Hi Rahul,
I have tested the migration of murine and human melanoma cells by the modified Boyden Chamber under different concentrations of a tested agent, i think that you used the same concentration of the chemo-attractant (in 150ul media 1%BSA) in the top and bottom of the insert. there must be a difference in concentration like the bottom one is upper than that of the insert so that the cells can be attracted in this direction.
protocol
B16F1 melanoma invasiveness issued from resected tumour
was measured in vitro using Transwell® chambers (Sigma-Aldrich,
Saint Quentin, Fallavier, France) coated with Matrigel® (30 μg cm2,
BD Biosciences, France). Briefly, the in vitro invasion tests were
performed as follows: 50 000 tumour cells were suspended in
serum-free DMEM containing 0.2% bovine serum albumin (BSA)
and seeded on coated-membranes. DMEM supplemented with 2%
FBS and 2% BSA was used as a chemoattractant. After an
overnight incubation, cells were fixed with methanol and stained
with crystal violet. Cells remaining on the upper side of the
membranes were eliminated, and those on the lower side counted.
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