Cell Permeabilization and Exocytosis Assay

Examine the cells under the microscope. Be sure that all buffers are ready and equilibrated to 37°C. Test the waterpump aspiration should be as gentle as possible. It is good practice to test the aspiration on a microwell filled with water (without cells). The end of the tubing should be fitted with a yellow pipette tip. Aspiration and addition of solutions should always be done at the same point of the well, for example at 12 o clock . If cells are well attached you may briefly touch the bottom with the aspiration tip and pipette tip. We use 100 (i.1 for washes, 50 [j,l for all subsequent steps. If an agent to be tested is 

Set the timer and have the protocol in a place where you can easily see it. You will have little time to rectify any mistakes I 

Aspirate the medium carefully. Wash the cells with KRH, 100. 1/ well. If done in a regular manner, this does not require timing. 

Set the timer and start permeabilization by aspirating the KRH from the wells and adding 50 [il of SLO solution/well. 

After 7 min, aspirate the supernatant gently and add 50. 1 of preincubation or incubation solution. 

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