Ultracentrifugation-Based Isolation of Legionella pneumophila Outer Membrane Vesicles

Abstract

Source: Jung, A. L., et al. Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages. J. Vis. Exp. (2017)

This video demonstrates the isolation of outer membrane vesicles (OMVs) from Legionella pneumophila using sequential centrifugation, sterile filtration, and ultracentrifugation. The purified OMVs, enriched in membrane proteins and lipopolysaccharides, are suitable for host-pathogen interaction studies.

Protocol

1. Cultivate Legionella pneumophila

1. Spread L. pneumophila strain Corby (wild type, WT) on buffered charcoal yeast extract (BCYE) agar plates and incubate them at 37 °C for 3 days. Inoculate 10 mL of yeast extract broth (YEB) at an OD₆₀₀ of 0.3 with L. pneumophila from the preculture plate; incubate the bacteria at 37 °C on a rotating shaker (150 rpm) for 6 h.
2. Verify the purity of the liquid culture by spreading 100 µL of the suspension on a blood agar plate. Incubate overnight at 37 °C.
3. Add the remaining liquid culture to 90 mL of fresh YEB medium and incubate on a rotating sha....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm Petri dish	Sarstedt AG & Co KG (Nuembrecht, Germany)	82.1473
70 Ti rotor	Beckman Coulter Incorporation (California, USA)	337922
Cuvettes	Sarstedt AG & Co KG (Nuembrecht, Germany)	67.742
Heracell 240i CO₂ incubator	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)	40830469
Heraeus Multifuge X3R	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)	75004515
Inoculation loop	Sarstedt AG & Co KG (Nuembrecht, Germany)	86.1567.010
L. pneumophila Corby	---	---
PBS	Biochrom GmbH (Berlin, Germany)	L 1825
Rotating shaker (MaxQ 6000)	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)	SHKE6000
Ultrospec 10	Biochrom Ltd (Cambridge, England)	80-2116-30
Sterile filter (pore size: 0.22 µm)	Corning Incorporated (new York, USA)	431096
Activated charcoal	Carl Roth GmbH & Co KG (Karlsruhe, Germany)	X865.2
Agar-agar, Kobe I	Carl Roth GmbH & Co KG(Karlsruhe, Germany)	5210.2
Columbia agar with 5% sheepblood	Becton Dickinson GmbH(Heidelberg, Germany)	254005
Yeast extract	Carl Roth GmbH & Co KG(Karlsruhe, Germany)	2363.2