This article details a method for isolating extracellular vesicles (EVs) from Mycobacterium tuberculosis cultures grown in iron-limiting conditions. The process enhances the secretion of siderophore-containing EVs, which are important for downstream studies.
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 pipette and filter sterilize it through a 0.22 micron filter.
To isolate MEVs, transfer the culture filtrate into a stirred cell ultrafiltration system placed at four degrees Celsius and filter the concentrate at a pressure less than 50 PSI through a 100 kilodalton cutoff membrane. Then centrifuge the concentrated culture filtrate at 15,000 times g for 15 minutes at four degrees Celsius and collect the supernatant in polycarbonate ultracentrifugation tubes. Centrifuge the supernatant at 100,000 times g for two hours at four degrees Celsius.
After that, remove the supernatant and resuspend the membranous pellets in a total of one milliliter of sterile PBS by gentle pipetting. Mix 0.5 milliliters of the pellet suspension with 1.5 milliliters of 60% iodixanol solution at the bottom of a polypropylene thin walled ultracentrifuge tube. Overlay this MEV iodixanol 45% suspension with one milliliter of 40%, 35%, 30%, 25% and 20% iodixanol solutions and one milliliter of PBS at the top.
Centrifuge at 100,000 times g for 18 hours at four degrees Celsius. Next, use a one milliliter Hamilton syringe to collect the one milliliter density gradient fractions starting from the top. Dilute each collected fraction to 20 milliliters with PBS and centrifuge at 100,000 times g for two hours at four degrees Celsius.
Remove the supernatant and resuspend in 0.5 milliliters of PBS.
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