At 1 h post-infection, nocodazole was added to the culture medium to a ®nal concentration of 25 mM to depolymerize microtubules. At 2 h postinfection, the cells were washed 3±4 times in warm medium to remove nocodazole. Washed cells were incubated in medium without nocodazole for the indicated time at 37°C to re-initiate microtubule polymerization; they were then washed brie¯y in warm phosphate-buffered saline (PBS) and immediately ®xed. In parallel, samples were also removed at the same time point, brie¯y rinsed in ice-cold PBS, ®xed and processed for immuno¯uorescence to con®rm complete microtubule depolymerization before initiation of microtubule assembly. Uninfected control HeLa cells were treated and processed in an identical fashion. The same numbers of images were integrated using identical camera settings to allow direct comparison between infected and uninfected samples from the same experiment. The site of origin of the 1918 influenza pandemic and its public health implications  The 1918-1919 influenza pandemic killed more people than any other outbreak of disease in human history. The lowest estimate of the death toll is 21 million, while recent scholarship estimates from 50 to 100 million dead. World population was then only 28% what is today, and most deaths occurred in a sixteen week period, from mid-September to mid-December of 1918.