By EdithH,,Quaranta lab, Scripps
Transwell-Migration Assay
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Coat lower side of tissue-culture treated transwell inserts (Costar) o/n at 4C with ECM in
PBS (for HaCaT: 0.3-0.5ug/ml Ln-5; 1ug/ml col-IV; 10ug/ml FN, 15ug/ml Ln-1/2).
There are 12 inserts per one 24well plate. (The extra wells are for easy washing, see later*).
Use 75 ul per insert. Pipet solution on bottom of 24well plate well and immediately set insert
on top of the ECM drop. If you wait too long, the ECM drop starts to float around and you
loose ECM. Thus, coat one well after the other. Coat two inserts for each condition you plan
to test.
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Transfer inserts to empty wells*. Discard ECM solution and add 300 ul of PBST, transfer
inserts back into these PBST filled wells. Fill empty wells with 300 ul of PBST, transfer
inserts from “old” PBST filled wells into the freshly filled wells. These procedure = 2x
washed with PBST.
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Discard PBST, add 300 ul of 5% milk in PBST and transfer inserts. Incubate at RT until cells
are ready.
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During blocking reaction, prepare cells:
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Detach cells with trypsin, or better with EDTA, if possible. Add normal culture medium to
block trypsin, spin.
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Resuspend cells in serum-free culture medium, spin.
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Repeat above step.
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Resuspend cells in serum-free culture medium, count.
Dilute cells to 0.1-1.2 mio/ml (1.2 mio/ml okay for HaCaT).
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Aliquot cells into 2ml tubes e.g 250 ul/tube for each condition.
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Add antibodies (20-50 ug/ml final) or other reagents, incubate for 15 min at RT.
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Add antibodies (20-50 ug/ml final) or other reagents, incubate for another 15 min at RT.
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During incubation, prepare 1.5ml tubes containing 1.2 ml serum-free culture medium, one
tube for each condition. Add antibodies (20-50 ug/ml final) or other reagents. This is the
“lower chamber buffer” for the 24well plate.
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Discard milk and wash blocked inserts 3x with 300 ul PBS. Follow procedure described
above*.
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Transfer lower chamber buffer into wells, 600 ul per well. Remove PBS that might be in the
inserts and then transfer inserts into wells.
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Mix cells and seed 100 ul/insert. (Thus, 2 x 100 ul for each condition tested).
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Incubate at 37C for 5 hrs or longer.
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To fix cells, fill empty wells with 400 ul fixation solution (Hema kit, Fisher), remove medium
in inserts and transfer inserts into fixation solution. Incubate at RT for 10 min.
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Meanwhile, remove lower chamber medium and add 400 ul staining solution (kit). Remove
fixation solution in inserts and transfer inserts into staining solution. Incubate at RT for 20
min.
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Wash inserts 2x with water, following procedure described above*.
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Using a cotton swab, remove cells on upper side of the insert. Make sure you remove all the
cells since it is not possible to distinguish between cells on upper or lower side of the insert
under the microscope. Cells that have migrated from the upper side of the insert through the
pores to the lower side of the insert are counted = cells per microscopic field.
PBST: PBS + 0.2% Tween-20
E.H. 1998