Dear Colleagues
I am bout to explore the extravasation of tumours by in vitro modelling using a trans well (Boyden chamber) assay; I must confess this is completely new to me
In essence, we are coating the trans well insert with huvec cells and then seeding on a tumour line to investigate migration of tumour cells (through the Huvec monolayer) to the opposite side of the insert and also into the underlying medium. Specifically:
1. How do you count the cells that migrate through the huvec monlayer to the underside of the membrane and/or culture medium below ?: Recovering cells, staining and then counting manually or by recovering cells and counting by flow cytometry ? Which is considered best practice ?
2. How do you distinguish between tumour cells that have migrated through the Huvec layer and potentially Huvec cells that may have detached and also penetrated the insert ?: I though about just setting up some control cultures not seeded with Tumour lines, i.e. Huvec monolayer and only and attempting to gauge cells recovered from the opposite side of the insert and/or undelying culture medium (in well in which boyden chamber sits). Would this be sensible/informative ?
3. In the context of an assay like this is it considered good practice to count both cells recovered from the underside of insert itself as well as those that have migrated into the culture medium below ?
As you can infer from my line of questioning I am a complete novice to this set up so all suggestion no matter how simple are welcome !!
Much obliged
Hi Laurence,
Although, we have been using the trans-endothelial migration (TEM) assay to look at immune cell trafficking across primary endothelial cells and not tumour cells, the fundamentals should be the same (I hope!).
As you have already pointed out, there are a number of ways to quantify the number of migrating cells with the choice dependent on what experimental set up you want. For example, we are interested in medium throughput assays (96-well plates) so we use a DNA binding dye (CyQuant) to quantify total numbers of migrating cells or flow cytometry when the specific type or activation state of the migrating cells is required. If you only plan to run a few TEM samples at a time, then perhaps direct cell counting would be easier and more relevant.
As long as the endothelial monolayer hasn’t been kept in culture on the inserts for too long (we seed quite high and grow for 48hrs), then there should be limited growth of the cells through the pores. Your suggested control with no-tumour cells is a good one to check for this directly. Alternatively, you could pre-label the tumour cells with a cell tracker dye before adding to the inserts, to ensure that you only count the cell type you are interested in! From our experience, a significant number of migrated immune cells will still be attached to the underside of the insert following migration, so we routinely detach these cells and include them in our quantification. I’m unsure if this would apply to your tumour cells but it worth checking during the assay optimisation.
The selection of an appropriate chemokine to trigger migration of immune cells is critical in my assays, would the same be true for your tumour cell lines?
Good luck & let us know how you get on.
Mark
I have used Calcein AM to stain my tumor cells with fluorescent dye. My endothelial cells have also migrated, but Calcein AM gives very nice strong fluorescence so I could differentiate transmigrated tumor cells from migrated EC cells. I have used this dye despite my tumor cells expressing GFP. It was because tumor cells like to climb on migrated endothelial cells, so the bottom part of the filter was quite tri-dimensional. It makes for a great fluorescence background. You need very strong and uniform staining to observe tumor cells. Calcein AM gives it.
Hi Laurence, we usually use PKH26 fluorescent dye to stain the tumor cells in the upper chamber, and detect the fluorescent intensity using a plate reader. Different tumor cells have distinct migration capacities. In my case, I usually keep them culturing for 3-6 hours before reading. BTW, as you mentioned, you need to set a well with only cell monolayer in the bottom chamber as a negative control. What's more, you usually need to add some chemokine to trigger migration, or set a concentration difference between them, for example, no FBS in the upper chamber and 10% FBS in the bottom chamber.
Hope this information is helpful for you.
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