I am trying to do intracellular cytokine staining of macrophages to be read using flow cytometry. I have done it once with an incubation for 24 hours, yet when I was going to harvest, many cells were lifted, indicating that Brefeldin A had ben toxic to the cells, however the viability remained at 80%, as judged by flow cytometry using Zombie Aqua. I am going to try different incubation times below 24 hours, to be able to figure out the best time before the lifting happens is, yet I would like to know what other people have done to have a better guide that just trial and error.