E. coli MG1655 cells were grown overnight in LB medium and subsequently diluted 1000 times in EZ rich medium (Teknova) with 0.2% glucose. The cells were grown until the culture reached an OD600 value of 0.3 after which the cells were centrifuged at 3000 rcf for 5 minutes and resuspended in PBS buffer. The amino-reactive dye Cy3B-NHS (GE Healthcare) was added to a final concentration of 1 mM and incubated while protected from light at room temperature for 1 hour [89 (link)]. Next, the cells were centrifuged at 3000 rcf for 10 minutes and the cell pellet was resuspended in PBS.
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