Ultracentrifugation-Based Isolation of Legionella pneumophila Outer Membrane Vesicles

Samenvatting

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 shaker (37 °C and 150 rpm) to reach an OD₆₀₀ of 3.0-3.5, which takes approximately 16-20 h.

2. Prepare and Quantify L. pneumophila Outer membrane vesicles (OMVs)

NOTE: Carry out all of the following centrifugation steps under sterile conditions and at 4 °C.

1. Centrifuge the liquid culture at 4,000 x g for 20 min to pellet the bacteria. Transfer the supernatant to fresh centrifuge tubes, discard the bacterial pellet, and repeat the centrifugation (4,000 x g for 20 min). Repeat this step once.
2. Sterile-filter the remaining supernatant twice (pore size: 0.22 µm). Transfer the bacteria-free supernatant to ultracentrifuge tubes and ultracentrifuge at 100,000 x g for 3 h.
3. Decant the supernatant and discard it. Resuspend the OMV pellet in sterile phosphate-buffered saline (PBS) and ultracentrifuge (100,000 x g for 3 h) to remove contaminating proteins and LPS.
4. Discard the supernatant and resuspend the OMV pellet in 500 µL of sterile PBS. Streak 20 µL on a blood agar plate and on a BCYE agar plate to exclude bacterial contamination of the prepared vesicles. Incubate the blood agar plate overnight and the BCYE agar plate for 3 days (both at 37 °C).
   NOTE: The concentration of 100 mL of L. pneumophila culture is usually 1 µg/µL. Store the prepared and quantified OMVs at -20 °C.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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