Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Abstract

Source: Anderson, L. S., et al. A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection. J. Vis. Exp. (2019).

This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Mouse Source and Housing Derive LysM-eGFP mice on a C57BL/6J genetic background. Derive LysM-EGFP×MyD88-/- mice by crossing LysM-EGFP mice with MyD88-/- mice on a C57BL/6J background. House mice in a vivarium. For these studies, animals were housed at the University of Cali…

Representative Results

Figure 1: Correlation between OD600 and CFU counts for ALC2906. 3-4 ALC2906 SH1000 colonies were picked from an agar plate and transferred into TSB with 10 µg/mL chloramphenicol for overnight culture. The next day, the suspension was split 1:50 into TSB with 10 µg/mL chloramphenicol and cultured. Optical density at 600 nm (OD600) was measured in regular intervals after 2 hours using a sp…

Materials

14 mL Polypropylene Round-Bottom Tube	Falcon	352059
6mm Disposable Biopsy Punch	Integra Miltex	33-36
Bioluminescent S. aureus	Lloyd Miller, Johns Hopkins	ALC 2906 SH1000
Bovine Blood Agar, 5%, Hardy Diagnostics	VWR	10118-938
Buprenoprhine hydrochloride injectable	Western Medical Supply	7292	0.3 mg/mL
C57BL/6J Mice	Jackson Labratory	664
Chloramphenicol (crystalline powder)	Fisher BioReagents	BP904-100
DPBS (1X)	Gibco	14190-144
Insulin Syringes	Becton, Dickson and Company	329461	.35 mm (28 G) x 12.7 mm (1/2'')
IVIS Spectrum In Vivo Imaging System	Perkin Elmer	124262
Living Image Software – IVIS Spectrum Series	Perkin Elmer	128113
LysM-eGFP Mice	Thomas Graff Albert Einstein College of New York	NA
Microvolume Spectrophotometer	ThermoFisher Scientific	ND-2000
MyD88 KO Mice	Jackson Labratory	9088
Non-woven sponges	AMD- Ritmed Inc	A2101-CH	5 cm x 5 cm
Povidone Iodine 10% Solution	Aplicare	697731
Tryptic Soy Broth	Becton, Dickson and Company	211825