E. coli OMVs

Outer membrane vesicles (OMVs)

E. coli outer membrane vesicles - Caspase 11-4/5 inducers

InvivoGen provides two grades of E. coli outer membrane vesicles (OMVs) to foster your research on the non‑canonical inflammasome response:

— E. coli OMVs (standard preparation) - recommended for in vitro studies

— E. coli OMVs InvivoFit™ - recommended for in vivo studies.

OMVs are small, spherical bodies secreted by gram-negative bacteria. They were recently described as potent caspase 4/5 (caspase 11 in mice) inflammasome inducers [1].

 More details

InvivoGen's OMVs have been purified from Escherichia coli BL21 using an optimized procedure, providing lot-to-lot reproducibility. Importantly, activation of the caspase 11-4/5 non-canonical inflammasome is confirmed in vivo and using  THP1-HMGB1-Lucia™ cells. 

Key features:

• Inducer of non-canonical caspase 11-4/5 inflammasome pathways in vitro and in vivo
• Can be used in murine or human cellular assays
• Each lot is characterized (size) and functional tested (activation of TLR2 and TLR4).
• InvivoFit™ grade: guaranteed sterile, endotoxin level >10⁵ EU/mg

Read our review on the NLRP3 inflammasome.

Download our Practical guide on Inflammasomes.

1. Vanaja, S.K. et al., 2016. Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation. Cell 165:1106-1119.

Figures

Figure 1: E. coli OMVs were prepared for electron microscopy using conventional negative staining procedures. E. coli OMVs (20 μl) were absorbed onto Formvar carbon-coated grids for 2 min, blotted, and then negatively stained with 1% uranyl acetate for 1 min. Grids were then examined with a transmission electron microscope (Jeol JEM‑1400, JEOL Inc, Peabody, MA, USA) at 80 kV. Images were acquired with a digital camera (Gatan Orius, Gatan Inc, Pleasanton, CA, USA) using a range of magnifications (20 nm magnification seen to the left).

Figure 2: THP1-HMGB1-Lucia™ cells were incubated with 0.2 ng/ ml-20 μg/ml of E. coli OMVs for 6 (blue) and 24 hrs (red). After the incubation, Lucia luciferase activity was determined by measuring relative light units (RLUs) in a luminometer using the QUANTI-Luc™ detection reagent. Results are presented as normalized activity (percentage % RLUs).

Figure 3: Bone marrow-derived macrophages (BMDMs) from wild type (WT) (blue) and caspase-11 knockout (KO) (red) mice were treated with a range of different concentrations (from 0.5 to 2.5 μg/250 000 cells) of E. coli OMVs (prepared in PBS) for 16-20 hours. After the incubation, the cell supernatants were used for both a (A) lactate dehydrogenase (LDH) assay and an (B) ELISA for measuring the release of IL-1β.

Figure 4: Wild type (WT) (blue) and Gasdermin-D knock out (KO) (red) mice (20-25g) were injected with 150 μg of poly IC (LMW) for the priming stage of inflammasome activation and then left for 6 hours. Following this, a range of different concentrations from 10-50 μg of E. coli OMVs (prepared in PBS) were injected intraperitoneally into the mice. After an additional 5 hours, peritoneal (A) IL-1α and (B) IL-1β cytokine levels were assayed using ELISA kits.

Specifications

E. coli OMVs

Source: Escherichia coli BL21

Specificity:  Caspase 11-4/5 inflammasome

Working concentrations: : 0.2 - 100 µg/mL (in vitro) and 10 - 100 µg/mouse (in vivo)

Quality Control:

• Size range: 35-60 nm
• Activation of TLR2 and TLR4 by E. coli OMVs has been confirmed using HEK-Blue™  TLR cellular assays.
• Inflammasome activation by E. coli OMVs has been determined by the induction of pyroptosis in THP1-HMGB1-Lucia™ cells.

E. coli OMVs InvivoFit™

Source: Escherichia coli BL21

Specificity: Caspase 11-4/5 inflammasome

Working concentrations: 0.2 - 100 µg/mL (in vitro) and 10 - 100 µg/mouse (in vivo)

Quality Control:

• Endotoxin levels:  >10⁵  EU/mg of OMVs
• E. coli OMVs InvivoFit™ are guaranteed sterile.
• Size range: 35-60 nm
• Activation of TLR2 and TLR4 by E. coli OMVs InvivoFit™ has been confirmed using HEK-Blue™  TLR cellular assays.
• Inflammasome activation by E. coli OMVs InvivoFit™ has been determined by in vivo assays and the induction of pyroptosis in THP1-HMGB1-Lucia™ cells.

Contents

Please note: each ligand is sold separately.
The quantity of protein was measured by a BCA protein assay. Both ligands are provided with 1.5 ml of sterile, endotoxin-free water.

E. coli OMVs

• tlrl-omv-1: 100 µg

E. coli OMVs InvivoFit™

• tlrl-omv: 500 µg 

Product is shipped at room temperature

Upon receipt product should be stored at -20°C

InvivoFit™

InvivoFit™ is a high-quality standard specifically adapted for in vivo studies. InvivoFit™ products are filter-sterilized (0.2 µm) and filled under strict aseptic conditions in a clean room. The level of bacterial contaminants (endotoxins and lipoproteins) in each lot is verified using a LAL assay and a TLR2 and TLR4 reporter assay.

Details

OMVs are small, non-replicative, immunogenic spherical bodies secreted by both commensal and pathogenic gram-negative bacteria [1]. They contain various pathogen-associated molecular patterns (PAMPs) such as DNA, RNA, peptidoglycan, protein, toxins and lipopolysaccharides (LPS). These PAMPs can induce both pro- and anti-inflammatory responses; a balancing act that can result in either pathogen clearance or bacterial survival [2]. They interact with pattern recognition receptors (PRRs) expressed on the surface of host cells such as Toll-like receptors (TLRs). Additionally, OMVs can be endocytosed and deliver their highly immunogenic cargo directly to the cytosol of the host cell. This triggers the non-canonical caspase 11-4/5 inflammasome in mice and humans, respectively. Ultimately, the activation of this complex results in pyroptotic cell death through the action of gasdermin D (GSDMD) and the release of pro-inflammatory cytokines such as IL-1β and IL-18 [3].

References:

1. Kulp, A. & Kuehn, M.J., 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64:163-184.
2. Kaparakis-Liaskos, M. & Ferrero, R.L., 2015. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol 15:375-387.
3. Vanaja, S.K. et al., 2016. Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation. Cell 165:1106-1119.