The exocytosis rate was determined as described previously (13). Briefly, cells were preincubated at 37°C with saturating concentration of anti-HA antibody to label YFP-HA-Orai1 and fixed after various incubation times. Initially, Orai1 at the PM was labeled, and an increase in total cell-associated anti-HA antibodies was observed over time as more Orai1 trafficked to the PM and interacted with anti-HA antibodies. Cells were then fixed, permeabilized, and stained with Cy5-labeled anti-mouse antibody. Therefore, the time course of the Cy5/YFP ratio reflects the exocytosis of YFP-HA-Orai1. Data were fit to a monoexponential growth curve as discussed previously to determine the exocytosis rate (17).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.