Using Fluorescence-Mediated Tomography to Assess Murine Intestinal Inflammation

Abstract

Source: Nowacki, T. M., et al. Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation. J. Vis. Exp. (2017)

This video demonstrates a technique for assessing intestinal macrophage infiltration in a murine model of colitis using fluorescence-mediated tomography (FMT). Upon inducing colitis in a mouse via dextran sodium sulfate (DSS), the animal is injected with antibodies that recognize the F4/80 glycoprotein marker on infiltrated macrophages in the colon. The mouse is then imaged in an FMT device where the fluorescence and reflectance signals are reconstructed into a three-dimensional image to determine the spatial distribution and intensity of fluorescence.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Materials and Experimental Setup

1. Animal care.
   1. Use sex- and age-matched mice of any DSS-susceptible strain (e.g., C57BL/6) at 20-25 g body weight.
   2. Plan at least five or more mice per experimental group and house the mice according to local animal care guidelines.
   3. Provide a standard rodent chow diet and autoclaved drinking water ad libitum.
   4. Remove the standard chow and replace it with alfal....

Results

Figure 1. Tomographic visualization of macrophage infiltrates in murine colitis.

FMT scans in colitic mice and non-colitic controls administered Cyanine7-conjugated antibody against murine F4/80 (n = 5 per group). <.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Alfalfa-free diet	Harlan Laboritories, Madison, USA	2014
Bepanthen eye ointment	Bayer, Leverkusen, Germany	80469764
Dextran sulphate sodium (DSS)	TdB Consulatancy, Uppsala, Sweden	DB001
Eosin	Sigma - Aldrich, Deisenhofen, Germany	E 4382
Ethylenediaminetetraacetic acid (EDTA)	Sigma - Aldrich, Deisenhofen, Germany	E 9884
Florene 100V/V	Abbott, Wiesbaden, Germany	B506
Haematoxylin	Sigma - Aldrich, Deisenhofen, Germany	HHS32-1L
O.C.T. Tissue Tek compound	Sakura, Zoeterwonde, Netherlands	4583	fixative for histological analyses
Phosphate buffered saline, PBS	Lonza, Verviers, Belgium	4629
Sodium Chloride 0,9%	Braun, Melsungen, Germany	5/12211095/0411
Sodium bicarbonate powder	Sigma Aldrich Deisenhofen, Germany	S5761
Standard diet	Altromin, Lage, Germany	1320
Tissue-Tek Cryomold	Sakura, Leiden, Netherlands	4566
Hemoccult (guaiac paper test)	Beckmann Coulter, Germany	3060
Biotin rat-anti-mouse anti-F4/80 antibody	Serotec, Oxford, UK	MCA497B
Biotin rat-anti-mouse anti-GR-1	BD Pharmingen, Heidelberg Germany	553125
Streptavidin-Alexa546	Molecular Probes, Darmstadt, Germany	S-11225	excitation/emission maximum: 556/573nm
Anti-CD11b rat-anti-mouse antibody TC	Calteg, Burlingame, USA	R2b06
Purified anti-mouse F4/80 antibody	BioLegend, London, UK	123102
DAPI	Sigma-Aldrich, Deisenhoffen, Germany	D9542
FITC-conjugated anti-Ly6C rat-anti-mouse antibody	BD Pharmingen, Heidelberg, Germany	553104
FACS buffer	BD Pharmingen, Heidelberg, Germany	342003
Cy7 NHS Ester	GE Healthcare Europe, Freiburg, Germany	PA17104
MPO ELISA	Immundiagnostik AG, Bensheim, Germany	K 6631B
Cy5.5 labeled anti-mouse F4/80 antibody	BioLegend, London, UK	123127	ready to use labelled Antibodies (alternative)
Anti-Mouse F4/80 Antigen PerCP-Cyanine5.5	eBioscience, Waltham, USA	45-4801-80	ready to use labelled Antibodies (alternative)
DMSO (Dimethyl sulfoxide)	Sigma-Aldrich, Deisenhoffen, Germany	67-68-5
Isoflurane	Sigma-Aldrich, Deisenhoffen, Germany	792632
Ethanol	Sigma-Aldrich, Deisenhoffen, Germany	64-17-5
Bovine Serum Albumins (BSA)	Sigma-Aldrich, Deisenhoffen, Germany	A4612
Tris Buffered Saline Solution (TBS)	Sigma-Aldrich, Deisenhoffen, Germany	SRE0032
Equipment
FACS Calibur Flow Cytometry System	BD Biosciences GmbH, Heidelberg, Germany
FMT 2000 In Vivo Imaging System	PerkinElmer Inc., Waltham, MA, USA	FMT2000
True Quant 3.1 Imaging Analysis Software	PerkinElmer Inc., Waltham, MA, USA	included in FMT2000
Leica DMLB Fluorescent Microscope	Leica, 35578 Wetzlar, Germany	DMLB
Bandelin Sonopuls HD 2070	Bandelin, 12207 Berlin, Germany	HD 2070	ultrasonic homogenizer
Disposable scalpel No 10	Sigma-Aldrich, Deisenhoffen, Germany	Z692395-10EA
Metzenbaum scissors 14cm	Ehrhardt Medizinprodukte GmbH, Geislingen, Germany	22398330
luer lock syringe 5ml	Sigma-Aldrich, Deisenhoffen, Germany	Z248010
syringe needles	Sigma-Aldrich, Deisenhoffen, Germany	Z192368
Falcon Tube 50ml	BD Biosciences, Erembodegem, Belgium	352070