Transmigration assay - (Dec/14/2007 )
I have been trying to do transmigration assay with dendritic cells.I am using 5.0micrometer poresize inserts.I am really having a hard time standardizing the assay.If anyone has done this before, I would be thankful if you could help me with this.
Regards.
Regards.
you need a good number of independent experiments (n-number) with a high number of tests in transwell migration or invasion assay; we use 96-well plates testing 12-time the same test with at least 4 experiments;
hi seema,
u mentioned consequences but not the problem.
if u state the problem, then some one can come up with solutions.
in my opinion dendritic cells are bigger in size but the membrane u r using is with samll pores
(try with 8um filters. )
i have seen several reference for dendritic cell migration in 8um membrane but i do not have access to them.
gud luk
sravan.
Regards.
Seema,
Your poresize is fairly small for dendritic cells. I have used 5 um extensively for monocytes, and its almost impossible for it to work with endothelial cell types, so I would recommend higher pore size. Secondly, the type of membrane you use also matters. What membrane are you using? polycarbonate or nitro?
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I am using a polycarbonate filter. The cells are able to pass through the filter however, I am getting a lot of non specific migration in my medium control.I am also losing a lot of cells during the washing and scraping process [ to remove cells from the inner side of the insert].So the results I am getting are highly inconsistent. 
Do you have any suggestions?
u mentioned consequences but not the problem.
if u state the problem, then some one can come up with solutions.
in my opinion dendritic cells are bigger in size but the membrane u r using is with samll pores
(try with 8um filters. )
i have seen several reference for dendritic cell migration in 8um membrane but i do not have access to them.
gud luk
sravan.
The cells are able to pass through the filter however, I am getting a lot of non specific migration in my medium control.I am also losing a lot of cells during the washing and scraping process [ to remove cells from the inner side of the insert].So the results I am getting are highly inconsistent.
hi seema,
i m not practically familiar with inserts,
if possible, when u remove cells from non migrated surface just dip the membrane upper surface in the PBS and remove cells with a cell scraper.
regarding ur second question, after removing the cells, u can fix cells by airdrying, then stain with DAPI, this technique was implemented by me and used in one of my publications (payeli s.k., 2007, atherosclerosis) for THP-1 cells. this improvement avoid the loss of cells during fixation step, which i personally observed in my experiments.
gud luk