In Vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis Protocol

Experiment Summary

Bacterial lipopolysaccharide (LPS) is present in the outer membrane of Gram-negative bacteria and functions as pathogen-associated molecular pattern (PAMP). LPS therefore is a potent activator of inflammatory responses leading to cytokine release and neutrophils recruitment. The lipid A moiety of LPS activates the complex consisting of the LPS binding protein (LBP), CD14, MD-2 and Toll-like receptor 4 (TLR4) and the non-canonical inflammasome-linked caspases-4, 5 and 11, which in turn activate the canonical NLRP3 inflammasome. In particular, the cytokine interleukin (IL)-1β produced in response to inflammasome activation has a crucial role in neutrophil recruitment through promoting neutrophil adhesion and migration. This protocol allows studying of inflammatory response induced by LPS that affect neutrophil infiltration by tracking myeloperoxidase (MPO) activity in vivo.

Materials and Reagents

Insulin syringes
0.22 μm filters
C57BL/6 mice, typically of 8-12 weeks old mice (male or female)
LPS E.coli 0111:B4
LPS Extraction Kit
Dulbecco's phosphate-buffered saline (DPBS)
XenoLight RediJect inflammation probe
Luminol sodium salt
Isofluorane
5 mg/ml LPS
20 mg/ml luminol sodium salt stock solution

Equipment

Anesthesia machine or similar anesthesia equipment
Rechargeable trimmer
Scale
Biosafety cabinet
IVIS spectrum or a comparable luminescence imaging equipment

Software

Living Image software (PerkinElmer)

Procedure

Two days before the LPS intraperitoneal injection, place mice in the anesthesia machine and once the mice are anesthetized shave abdominal area with a trimmer, as fur quenches the luminescence signal (Fig. 1).

Fig. 1 A representative picture of mice under anesthesia getting their abdominal area shaved with a trimmer.

2. Weigh mice.

3. In the day of the experiment dilute LPS in DPBS and prepare syringes for injection.

4. Intraperitoneally inject mice with 2.5 mg/kg of LPS or the same volume DPBS for the control group (injection volume approximately 200 μl).

5. 3 h later intraperitoneally inject mice with 200 mg/kg of XenoLight Rediject inflammation probe or 200 mg/kg luminol sodium salt (injection volume approximately 200 μl).

6. Place mice in the IMPAC6 anesthesia chamber attached to the IVIS spectrum.

7. Transfer mice to the IVIS spectrum and place mice abdomen facing up into the chamber and position each nose inside the cone that delivers the isofluorane (Fig. 2).

8. Start imaging anesthetized mice 10 min post XenoLight Rediject inflammation probe injection with a 5 min exposure capturing in vivo bioluminescence generated by the activity of MPO as a marker for infiltration of neutrophils. In order to imagine 5 mice select field of view D and select 1.5 cm subject height (Fig. 2 and Fig. 3).

Fig. 2 A representative example of in vivo imaging in 8 wk of age male C57BL/6 mice after i.p. injection of PBS (left) or LPS (2.5 mg/kg body weight) (right).

Fig. 3 Screen capture image of the IVIS acquisition control panel.

9. Quantify the MPO signal using the Living Image software. First select region of interest (ROI) using ROI tools and choose to automatically draw measurement ROIs and perform ROI analyses to measure photon radiance. Also measure background ROI and subtract from your ROI measurement. Use average radiance to plot your graph.

* For research use only. Not intended for any clinical use.

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In Vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis Protocol

Experiment Summary

Materials and Reagents

Equipment

Software

Procedure

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