Intracellular Localization of Labeled Peptides in E. coli

The synthetic peptides were labeled with fluorescein isothiocyanate (FITC) by ChinaPeptides (QYAOBIO) Co., Ltd. (Shanghai, China). Intracellular localization of peptides was performed according to previous studies [47 (link),49 (link)]. As described in Section 3.8, E. coli was cultured to reach a viable bacteria count of approximately 10⁷ CFU/mL. The FITC-labeled peptides were dissolved in double-distilled water or dimethyl sulfoxide (final concentration 0.25%) at a final concentration of 250 μg/mL. Two peptides were mixed in equal proportions at equal concentrations to obtain a final concentration of 125 µg/mL. E. coli was treated with the FITC-labeled peptides and incubated at 60 r/min and 37 °C for 3 h. The bacteria were collected by centrifugation and washed twice with 0.1 M PBS (pH 7.4) and the supernatant was discarded. The resulting bacteria were fixed with 4% paraformaldehyde for 30 min and centrifuged at 2000× g for 3–5 min. Then, the supernatant was discarded and the resulting bacteria were mixed with 400 μL DAPI and 100 μL PBS, left for 10–15 min in the dark, washed twice with PBS and resuspended. One drop of the bacterial solution was added to a microscope slide. After the mounting medium (G1401, Servicebio, China) treatment, LSCM (Nikon Eclipse Ti, Tokyo, Japan) was used to analyze the localization of the peptides in E. coli cells.