Alexa fluor 647 nhs succinimidyl ester

We used a Nikon Eclipse Ti2-E inverted fluorescence microscope with a ×100 (N.A. 1.40) oil-immersion objective for imaging DU885 electron-multiplying charge-coupled device camera (Andor) for imaging (Rojas et al., 2018 (link)). Devices were kept at 37°C for imaging. Cells were streaked out on LB agar containing 50 µg/mL hygromycin and incubated at 37°C for 2–3 d. Single colonies were inoculated into Middlebrook 7H9 containing 50 µg/mL hygromycin and incubated for ~48 hr at 37°C. The cells were then back diluted and added to the B04A microfluidic perfusion plate (CellASIC) during exponential phase. Plates were loaded with medium pre-warmed to 37°C. Cells were loaded into the plate, which was incubated at 37°C, without shaking, for 30 min before imaging. Medium was exchanged using the ONIX microfluidic platform (CellASIC). In the case where cells were stained with RADA, a TAMRA-based fluorescent d-amino acid (Tocris Bioscience), 1 µM RADA was added to the culture upon back dilution. If the cells were not stained with RADA, Alexa Fluor 647 NHS succinimidyl ester (Thermo Fisher Scientific) was added to the media as an occlusion dye (it is not cell wall permeable and thus can be used to track the cells). Cells were perfused with Middlebrook 7H9 medium for 5 min and then hyperosmotically shocked with 7H9 + 3 M sorbitol for 10 min.