Cell attachment and spreading may be improved by pre-incubating permeable supports in medium prior to seeding. On first use, try bracketing a range of seeding densities for optimum growth. Cells grown on permeable supports are often sensitive to initial seeding density for good cell attachment. 
Larger pore sizes may permit some cell types to migrate through the pores on the permeable support. Cell morphology and cell densities on permeable supports are influenced by filter pore size. The wetted, more rigid, cellulosic filter will serve as a support for the collagen-coated membrane. A wetted cellulosic membrane filter should be placed in direct contact with the underside of the Transwell insert membrane before it is cut out with a scalpel. The collagen-coated PTFE membrane is fragile and requires careful handling during removal. The polycarbonate or polyester membrane with the fixed and stained cells attached may be removed from the Transwell insert by carefully cutting around the membrane edges with a scalpel. A protocol, Trypsinization Procedure for Corning® Transwell® Inserts, for removing cells from Transwell inserts is available in the technical section of the Corning Life Sciences website. Then the dissociating solution should be added to both the well and the Transwell insert and incubated until the cells begin to come off. If it is necessary to remove cells from Transwell membranes, we recommend rinsing both the Transwell insert and the plate well. Protocols for fixing and staining Transwell® inserts are available in our online technical library. #TRANSLUCENT SYNONYM SERIES#
Processing steps may be carried out by sequentially moving the Transwell insert through a series of multiple well multiwell plate wells containing the appropriate reagents. Avoid using solvents that dissolve polystyrene, polycarbonate, or polyester membrane materials.
Cell monolayers may be fixed and stained while in the Transwell insert using standard cytological techniques. Transwell inserts have three openings for standard pipet tips to allow samples to be added or removed from the lower compartment. The medium level should be checked periodically and fresh medium added as required. The cells are then added in fresh medium to the Transwell insert and returned to the incubator. 
The plate should then be incubated for at least one hour or overnight at the same temperature that will be used to grow the cells.
An initial equilibrium period may be used to improve cell attachment by adding medium to the multiple well plate well and then to the Transwell insert. Recommended medium volumes are shown in the table below. Transwell inserts are used by first adding medium to the multiwell plate, followed by adding the Transwell inserts, and lastly adding the medium and cells to the inside compartment.
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