Biomag carboxyl magnetite nanoparticles

To isolate intracellular BCVs, bacteria were conjugated with BioMag carboxyl magnetite nanoparticles (Bangs Laboratories, BM570) to generate ''magnetic bacteria'' (Miao et al., 2016) . Briefly, 500 mL of magnetic nanoparticle stock solution was diluted in 10 mL of water, centrifuged at 1,000 3 g at 4 C for 30 s, and 9 mL of the supernatant was removed. Then, the particles were washed twice with 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer (Coolaber, SL3300), pH 5.2, and 40 mg of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDAC, Sigma, 341,006) was added to the suspension at room temperature for 30 min to activate the particles. The activated and washed particles were mixed with 5 3 10 9 live bacteria and incubated at 37 C for 30 min. After being blocked with 1% BSA at 37 C for 30 min, the magnetic bacteria were incubated with 5637 cells at an MOI of 100:1 for 30 min. Then, the infected cells were washed with PBS 3 times and treated with 100 mg mL À1 gentamycin for an additional 30 min. Then, the cells were scraped and lysed by repeated passaging through a 29-gauge needle (BD, 328421). The debris of host cells were removed by centrifugation at 2,000 3 g at 4 C for 5 min. The BCVs in the supernatant were harvested at 4 C using a magnetic cell separation rack (Invitrogen), then washed with cold PBS twice.