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Biomag carboxyl magnetite nanoparticles

Manufactured by Bangs Laboratories

BioMag carboxyl magnetite nanoparticles are superparamagnetic iron oxide nanoparticles (SPIONs) with carboxyl surface functionalization. They have an average diameter of 50-300 nanometers and possess a magnetite (Fe3O4) core.

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3 protocols using biomag carboxyl magnetite nanoparticles

1

Isolation of Bacterial Containing Vacuoles

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To isolate BCVs, UPEC cell were conjugated with BioMag carboxyl magnetite nanoparticles (BM570, Bangs Laboratories), and the magnetic bacteria were added to 2×107 BECs at different MOI (WT: 300:1; ΔmsbB mutant: 100:1 to ensure initial bacterial load in different strain infected BECs were comparable). One hour later, the plasma membrane of infected cells were mechanically disrupted by passage of the cell suspension through Gauge30 needles. The BCVs were isolated from the supernatant on a magnetic cell separation rack (BD IMagnet™, BD Biosciences, 552311).
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2

Isolation and Analysis of Intracellular BCVs

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To isolate intracellular BCVs, UPEC cell were conjugated with BioMag carboxyl magnetite nanoparticles (BM570, Bangs Laboratories) to generate “magnetic bacteria”. For this purpose, the particles were first washed twice in 0.1 M MES buffer (2-(N-morpholino) ethanesulphonic acid, pH 5.2), followed by activation with 4 mg EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide). The activated and washed particles were mixed with 0.5×109 live E.coli, and incubated for 30 min at 37°C. After blocking with 1% BSA, the magnetic bacteria were added to 2×107 BECs at different MOI (WT: 300:1; ΔmsbB mutant:100:1 to ensure initial bacterial load in different strain infected BECs were comparable). One hour later, the infected cells were scrapped and the plasma membrane was disrupted by repeated passage of the cell suspension through Gauge30 needles. The BCVs in the supernatant was then placed on a magnetic cell separation rack (BD IMagnet™, BD Biosciences, 552311) for washing and purification at 4°C. The presence and absence of specific interested molecules on purified BCVs was analyzed after SDS-PAGE and western blotting. The amount of Rab27b, the marker for BCVs was used to confirm similar amount of BCVs were isolated from different BECs or BECs infected with distinct strains.
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3

Isolation of Intracellular Bacterial Compartments

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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.
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