Why do the cells sometimes detach from the coverslip when an ibidi Culture-Insert is removed?

Here are some possible reasons to explain cell detachment:

• The cell density is too high. --> Seed fewer cells (the cell layer should be grown to 100% confluence after 24 hours, but not overgrown). Please see Application Note 21 "Wound Healing Assay (PDF) for a detailed protocol of a wound healing assay.

• The cells are starving. --> Increase the medium volume per insert well, or exchange the medium during cell growth.

• In rare cases, the cell type does not adhere well to the ibiTreat surface. --> First, coat the surface of the coverslip to promote cell attachment. Then, let the coating dry before inserting an ibidi Culture-Insert. Tips for coating can be found in Application Note 08 "Coating Protocols for ibidi Labware" (PDF).

Here are some additional tips for using the ibidi Culture-Inserts with a coating, in order to promote cell adherence:

• We highly recommend testing, in advance, whether or not removing an ibidi Culture-Insert will disrupt the coating in the migration gap. This can be analyzed by using a fluorophore-tagged antibody against the coating protein. If the fluorescence signal from the coating in the region of the migration gap is as homogeneous and intense as it is in the open wells, then it can be assumed that the coating will stay intact following insert removal. After several successful tests, it is generally safe to perform the actual experiment.

• If insert removal disrupts the majority of the coating, coat only the wells of the insert and then seed the cells. After insert removal, fill the gap by adding a low concentration of coating to the medium and incubate for approximately 30 minutes. Afterwards, replace the coating solution with normal medium and continue with the migration assay.

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