T cell transwell migration

Transwell migration assay

Scott B. Thompson and Jordan Jacobelli

Transwell assay media: RPMI supplemented with 2% (wt/vol) BSA and 10mM HEPES buffer. Filter sterilize and store at 4C.

For lymphocytes, particularly naïve cells, a 3um or 5um pore size is recommended. It is recommended to setup each condition in duplicate transwells.

If testing trans-endothelial migration, Bend.3 endothelial cells must be plated on the transwell inserts 2 days before the assay. To plate Bend.3 cells treat transwell inserts with 100uL of a 0.1% Gelatin solution for 1-2h at 37C (this is not strictly required but helps the Bend.3 cells adhere and form a more homogeneous monolayer), then wash once with PBS and plate 50x10^3 Bend.3 cells in each transwell insert (typically use 100uL of a 0.5x10^6 cells/mL suspension in Bend.3 DMEM media). Add 600uL of DMEM media to the bottom well. ~18h before the assay TNFa can be added to activate Bend.3 cells, to do this gently remove the media from the Bend.3 cells and switch it (in top and bottom wells) with DMEM media containing TNFa. Just before the assay carefully remove the media from the Bend.3 cells and transfer the transwell insert to a new well with assay media, then very gently wash out 2 times the Bend.3 cell monolayer using assay media (make sure not to disrupt the monolayer), finally add 100uL of assay media to the insert and let sit for a few minutes.

Prepare the transwell assay plate by adding 600uL of assay media with or without chemokines in the bottom wells of the transwells according to your experimental plan. Check that the empty transwell inserts do not leak by waiting a few seconds ad checking if the top chamber fills up with media, if it leaks transfer the media back into the bottom chamber and discard insert and try a new one. For transwells with Bend.3 monolayers keep these in a separate well (as described above) and only put them in the assay wells just before starting the assay.  Setup a couple of control wells (without transwell inserts) in which a known quantity of lymphocytes is added to 600uL of media, typically use 20% of the input cells. This will serve to normalize the cell counts and get actual percentages of transmigrated cells.

Lymphocytes may also be fluorescently labeled to compare a control and an experimental cell population migrating through the same transwell to minimize environmental variability. To do this, label your two cell populations with 1 μM CFSE or 10 μM CMTMR in RPMI with nothing added and incubate ~25 min at 37C, then wash 2-3 times in RPMI with 10% FCS. Count cells and mix them at a ratio of 1:1 to use for the transwell migration assay. It is recommended to also repeat the experiment switching the fluorescent dye labeling of the two populations.

Resuspend lymphocytes at 5-10x10^6 cells/ml in assay media and plate 100uL in the top chambers of the transwells. For transwells with Bend.3 monolayers, just before starting the assay transfer the transwells to assay wells with media in the bottom chamber. Immediately remove the media in the top chamber and add your lymphocyte suspension to the transwell.

Gently transfer your assay plate in the incubator and allow cells to migrate for 1-4h at 37C. A good portion of the migration happens within 1 hour but depending on your experimental conditions you may want to extend the assay up to 4 hours. The longer you incubate the more cells will migrate but more cells may also die since the assay media has no FCS. Furthermore, at 4 hours the migration may have plateaued and this may mask differences between your experimental conditions.

After the incubation, discard the transwell insert and collect the migrated lymphocytes by gently pipetting up and down a defined number of times for each well (we typically do 15) to dislodge adhered cells and transfer 300uL to a FACS tube on ice for analysis. Quantify the number of transmigrated cells for a fixed period of time (i.e. 2–3 min) using a Flowcytometer. Standardize your counts by transferring and acquiring 300uL of cell suspension from your control wells for the same amount of time as the other samples.

More recently we have also used cell counting beads to normalize the cell counts by adding a known number of beads to the experimental wells and the 20% input control wells. This can help if the flow stream on the Cytometer is not constant (e.g. if a partial clog were to happen).