This video demonstrates the pre-treatment of macrophages with Legionella pneumophila outer membrane vesicles (OMVs) to examine their effect on bacterial replication. By altering host immune signaling, OMVs make macrophages more permissive to infection. Higher colony counts in treated cells, compared to untreated ones, indicate that OMVs promote L. pneumophila replication inside macrophages.
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Method Article
Assaying Legionella pneumophila Replication in Macrophages Pretreated with Outer Membrane Vesicles
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August 29th, 2025
In This Article
Abstract
Protocol
1. Pre-treat Macrophages
- Prepare THP-1 (Tohoku Hospital Pediatrics-1 cells, a human monocytic leukemia cell line) cells.
NOTE: THP-1 is a monocytic cell line derived from a leukemia patient.- Add 2x105 THP-1 cells per 24 wells and differentiate them by adding 20 nM phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells. Incubate for 24 h at 37 °C.
- Replace the medium with 500 µL of fresh medium and incubate for another 24 h; the optimal medium for THP-1 cells comprises Roswell Park Memorial Institute Medium (RPMI) 1640 high glucose supplemented with 10% fetal calf....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 10 cm petridish | Sarstedt AG & Co KG (Nuembrecht, Germany) | 82.1473 | |
| 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 % sheep blood | Becton Dickinson GmbH (Heidelberg, Germany) | 254005 | |
| Ferric nitrate nonahydrate | Carl Roth GmbH & Co KG (Karlsruhe, Germany) | 5632.1 | |
| Fetal calf serum (FCS) | Life Technologies GmbH (Darmstadt, Germany) | 10270-106 | |
| Heracell 240i Carbon dioxide 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 | --- | --- | Kindly provided by Prof Dr Antje Flieger (RKI, Berlin, Germany) |
| L. pneumophila Corby ΔflaA | --- | --- | Kindly provided by Prof Dr Klaus Heuner (RKI, Berlin, Germany) |
| mBMDM | --- | --- | Kindly provided by Prof Dr Markus Schnare (Philipps Univeristy Marburg, Marburg, Germany) and Prof Dr Carsten Kirschning (University Duisburg Essen, Essen, Germany) |
| PBS | Biochrom GmbH (Berlin, Germany) | L 1825 | |
| Phorbol 12-myristate 13-acetate | Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) | P8139-1MG | |
| Rotating shaker (MaxQ 6000) | Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany) | SHKE6000 | |
| RPMI 1640 high glucose | Life Technologies GmbH (Darmstadt, Germany) | 11875-093 | |
| Saponin | Carl Roth GmbH & Co KG (Karlsruhe, Germany) | 9622.1 | |
| Ultrospec 10 | Biochrom Ltd (Cambridge, England) | 80-2116-30 | |
| THP-1 | Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) | 88081201-1VL | |
| Sorvall Discovery 100 SE | Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany) | ||
| Yeast extract | Carl Roth GmbH & Co KG (Karlsruhe, Germany) | 2363.2 |
