Isolation of Siderophore-Containing Extracellular Vesicles from Mycobacterium tuberculosis

Take a Mycobacterium tuberculosis culture grown in an iron-limiting medium to enhance the secretion of extracellular vesicles containing siderophores, an iron chelator.

Centrifuge to pellet the bacteria.

Collect the supernatant and filter it to remove residual bacteria.

Transfer the filtrate to a stirred ultrafiltration system and filter to remove excess media and concentrate the EVs.

Centrifuge the concentrate to pellet cellular debris.

Transfer the supernatant to an ultracentrifuge tube and centrifuge to collect the EV pellet.

Discard the supernatant, resuspend the pellet in a buffer, and mix it with a concentrated density gradient medium to form the bottom layer.

Overlay with decreasing concentrations of the gradient medium to create a discontinuous density gradient.

Add buffer at the top and centrifuge. The EVs migrate to the layer that matches their buoyant density.

Collect each fraction in a separate tube.

The purified fraction with siderophore-containing EVs is ready for downstream studies.

Transfer the culture to five 225 milliliter conical centrifuge tubes and centrifuge at 2,850 times g for seven minutes at 20 degrees Celsius. Collect the culture supernatant with a 50 milliliter pip