Isolation of Siderophore-Containing Extracellular Vesicles from Mycobacterium tuberculosis

Abstract

Source: Gupta, S., Marcela Rodriguez, G. Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria. J. Vis. Exp. (2019)

This video demonstrates the stepwise isolation of extracellular vesicles (EVs) from Mycobacterium tuberculosis cultured under iron-limiting conditions that mimic the host environment. It outlines the use of ultrafiltration, differential centrifugation, and density gradient separation to enrich and identify EV-containing fractions.

Protocol

1. Collection of Mycobacterial Extracellular Vesicles (MEVs)

1. Measure the OD₅₄₀ of the 2 mL culture at the time of collection. Make 1:10 serial dilutions of the culture and plate 100 µL of each dilution on agar plates with ADN and 0.05% Tween-80.
2. Transfer the culture to five 225 mL conical centrifuge tubes and centrifuge at 2,850 x g for 7 min at 20 °C.
3. Collect the culture supernatant with a 50 mL pipette and filter-sterilize it through a 0.22 µm filter unit.

2. Isolation of MEVs

1. Transfer the culture filtrate into a stirred cell ultrafiltration system placed at 4 °C and filter the concentrate at <50 psi through a 100 kDa cutoff membrane to ~50 mL.
2. Centrifuge the concentrated culture filtrate at 15,000 x g for 15 min at 4 °C and collect the supernatant.
3. Centrifuge the culture filtrate in polycarbonate ultracentrifugation tubes at 100,000 x g for 2 h at 4 °C.
4. Resuspend the membranous pellets in a total of 1 mL of sterile phosphate-buffered saline (PBS) by gentle pipetting.
5. Mix 0.5 mL of the pellet suspension obtained in step 2.4 with 1.5 mL of 60% iodixanol solution, yielding a final iodixanol concentration of 45% wt/vol. Dispense this mix at the bottom of a 13 mm x 51 mm polypropylene thin-walled ultracentrifuge tube.
6. Overlay the MEV-iodixanol 45% suspension with 1 mL of 40%, 35%, 30%, 25%, and 20% (vol/vol in PBS) iodixanol solutions and 1 mL of PBS at the top.
7. Centrifuge at 100,000 x g for 18 h at 4 °C.
8. Collect the 1 mL density gradient fractions starting from the top using a 1 mL Hamilton syringe.
9. Dilute each collected fraction to 20 mL with PBS and centrifuge at 100,000 x g for 2 h at 4 °C.
10. Remove the supernatant and suspend the pellet in 0.5 mL of PBS. Store this pellet at 4 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amicon stirred cell Model 108	EMD Milipore	UFSC40001	Cell Ultrafiltration system
BD Polypropilene 225 ml conical tubes	Fisher	05-538-61	Conical centrifuge tubes
Biomax 100-kDa cut-off ultrafiltration membrane	EMD Milipore	PBHK07610	Ultrafiltration membrane
Optiprep	Sigma	D1556	Iodixanol
Polycarbonate ultra centrifugation tubes 25 x 89 mm	Beckman Coulter	355618	Polycarbonate ultra centrifugation tubes 25 x 89 mm
Polypropylene thin walled centrifuge tube 13x15 mm	Beckman Coulter	344059	Polypropylene thin walled centrifuge tube 13x15 mm
Vacuum filtration flasks	CellPro	V50022	Filter Unit