Permeabilization cell seeding

Cells are seeded in a microwell plate 2 to 3 days before the experiment to reach about 80 % confluency on the day of the experiment to favour adherence during subsequent manipulations. Remember that certain cell lines have to be cultured on a special matrix. On the day of the experiment cells in a microwell plate are transferred to a 37°C waterbath. The temperature should be checked with a thermometer since variations may influence exocytosis. Cells are first washed with KRH, than permeabilized with SLO in 0.1 iM free Ca ". The cells are exposed to SLO for 7 min. Subsequently, cells are preincubated (if necessary) at 0.1 xM Ca and then shifted to 0.1 M or 10 jxM Ca ". Finally, the supernatant is transferred to microcentrifuge tubes, centrifuged at 10 000 g for 2 min to sediment any contaminating cells thereafter aliquots are used for determination of the secretory product. 

The seeding of cells should be performed at least 36 hr before the import assay. Place up to 12 coated coverslips, coated side facing up, into a 10-cm tissue dish. Trypsinize cells from a confluent dish and seed at 1 X 10 cells/lO-cm dish for permeabilization with 0.2 U/ml SLO and at 3 X 10 cells/lO-cm dish for permeabili-zation with 2.0 U/ml. Avoid cell clumping because it impairs the permeabilization with SLO. Two hours before the import assay the medium should be changed. 

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