I would like to confirm that my lymphocytes are stocked on top of the membrane of the transwell insert after a migration assay.
After the migration assay. I stained the transwell inserts with crystal violet in order to see the cells that remain in the membrane. I let the membranes to dry overnight.
Now I want to do a CD3-FITC staining of the membranes of the insert, to see that those cells are indeed T-cells. But the staining is very mild and difficult to see under a microscope. I have a few ideas of how to proceed, but I would very muhc appreciate your inputs:
I'll try to do CD3 staining on the membranes before the crystal violet, so therefore before the membrane is dry. (Dry tissue will not be too good for IHC)
Could I remove the membrane from the insert without damaging the cell layer? In that way, I could mount it on a coverslip and it would be easier to visualize under the microscope. Also, it would prevent the tissue from drying.
Right now I am incubating the Ab at 10ug/ml (recommended) for 1h RT.
Thank you in advance for your ideas and comments!
Alba
I think it is better to resuspend the cells from the membrane. T cells do not stick to the membrane so this is possible. Just carefully pipet up and down several times with buffer or medium above the membrane, and then you can take up the cells and transfer to tubes or plates for CD3 staining for flow cytometry.
Thanks for your answer Jolien.
I already do that, but when I count the cells fom the upper and lower chambers, the total number is about half of the cells I add to the upper chamber at the beginning of the experiment.
I plate a monolayer of endothelial cells on the membrane, through which the T-cells should go through. So my first guess is that the cells that I am missing are somehow trapped in this monolayer of endothelial cells nad the membrane.
Do you have any thoughts about that?
This is a little outside my area...
I was going to suggest the same thing as jolien because if you use trypsin, you should be able to get all cells off of the membrane and upper layer to run through the flow cytometry. Of course there is a risk of damaging and loosing cells too.
Can you use a 3-D matrix gel instead of transwell to do your assay for migration.
I am not too familiar with this technique but you can put your chemokine on the bottom of the matrix solution and have the T cell migrate through..you can fix the entire matrix (say like matrigel) to then slice it for imaging...
I am not sure anyone has done this.. but I do not see why you couldn't put chemokine on the bottom, matrigel, layer epithelial cells,matrigel, and then put T cells on the top.. a more "in vivo" setting.. then fix in paraformaldehyde, slice and stain.
Spontaneous suggestion outside my area..but maybe it will help brainstorm
good luck
Hi, can you please tell me which size inserts did you use to get this strong monolayer attached to your transwell, is it 1.0 µm 0r 0.4 µm pore?
Dear Najla,
I use 24well transwell plates of 8µm pore size, from corning.
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