Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.
Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.
Animal models are widely used in scientific research, and many non-invasive procedures exist to monitor disease activity and vitality, such as the quantification of body weight changes or the analysis of blood, urine, and feces. However, these are only indirect surrogate parameters that are also subject to inter-individual variability. They must frequently be complemented by post mortem analyses of tissue specimen, which prevents serial observation at repetitive time points and direct observation of physiological or pathological processes in vivo. Sophisticated small-animal imaging techniques have emerged, including cross sectional imaging, optical imaging, and endoscopy, which enables the direct visualization of these processes and also allows for repetitive analyses of the same animals1,2,3. Additionally, the possibility to repetitively monitor various states of disease in the same animal might decrease the number of animals needed, which might be desirable from an animal ethics point of view.
Several different optical imaging techniques exist for in vivo fluorescence imaging. Originally, confocal imaging was employed to study surface and subsurface fluorescent events4,5. Recently, however, tomographic systems that allow for quantitative three-dimensional tissue assessments have been developed6. This has been accomplished through the development of fluorescent probes that emit light in the near-infrared (NIR) spectrum, offering low absorption, sensitive detectors, and monochromatic light sources7. While traditional cross-sectioning imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), rely mostly on physical parameters and visualize morphology, optical imaging can provide additional information on underlying molecular processes using endogenous or exogenous fluorescent probes8.
Advances in molecular biology have helped to facilitate the generation of smart and targeted fluorescent molecular probes for an increasing number of targets. For example, receptor-mediated uptake and distribution in a given target area can be visualized using carbocyanine derivative-labeled antibodies9. The abundance of available antibodies, which can be labeled to function as specific tracers in otherwise inaccessible areas of the body, provides unprecedented insights into molecular and cellular processes in models of tumorigenesis and neurodegenerative, cardiovascular, immunologic, and inflammatory diseases7.
In this study, we describe the use of fluorescence-mediated tomography in a murine model of colitis. Dextran sodium sulfate (DSS)-induced colitis is a standard chemically induced mouse model of intestinal inflammation that resembles inflammatory bowel disease (IBD)10. It is particularly useful to assess the contribution of the innate immune system to the development of gut inflammation11. Since the recruitment, activation, and infiltration of monocytes and macrophages represent crucial steps in the pathogenesis of IBD, visualization of their recruitment and the kinetics of infiltration are essential to monitoring, for example, the effect of potential therapeutic substances in a preclinical setting12. We describe the induction of DSS colitis and demonstrate the tomography-mediated characterization of macrophage infiltration into the gut mucosa using fluorescence molecular tomography for the specific visualization of the monocyte/macrophage marker F4/8013. Additionally, we illustrate auxiliary and supplemental procedures, such as antibody labelling; the experimental setup; and analysis and interpretation of the obtained images, in correlation with conventional readouts such as disease activity indices, flow cytometry and histological analysis, and immunohistochemistry. We discuss limitations of this technique and comparisons to other imaging modalities.
All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen according to the German Animal Protection Law (Tierschutzgesetz).
1. Materials and Experimental Setup
2. Technical Equipment
3. Animal Anesthesia
4. Fluorescence-mediated Tomography Scan
NOTE: Adapt the following details, which are specific to the FMT system used in this study (see the Table of Materials) for alternative fluorescence reflectance imaging devices or FMT systems, as needed.
5. Post-scan
6. Data Reconstruction and Interpretation
7. Ex Vivo Analyses
Assessment of Colitis:
DSS-induced colitis is a chemically induced murine model of intestinal inflammation that resembles human IBD and leads to weight loss, rectal bleeding, superficial ulceration, and mucosal damage in susceptible mice15. It is particularly useful to study the contribution of the innate immune system to the development of intestinal inflammation10,11. To potently induce colitic inflammation, mice were continuously administered DSS throughout the experiment. Decreasing body weight and fecal occult blood were used as clinical indices of inflammation. As shown in Figure 1A, mice began losing weight starting at day four after the induction of colitis, whereas control animals receiving water alone showed no significant changes in body weight. In addition, colitic animals showed increased excretion of occult blood with feces (Figure 1B), as assessed using the following hemoccult scoring system: 0 (no blood), 1 (hemoccult positive), 2 (hemoccult positive and visual pellet bleeding), and 4 (gross bleeding, blood around anus)16. Colon length was measured to evaluate inflammation-induced colonic shortening, revealing significantly shortened colons in colitic mice as compared with control mice receiving water alone (Figure 1C). To directly assess colonic damage, histological analysis was performed at the end of the experiment. A quantitative assessment of histological damage was performed on colonic H&E-stained sections using an overall injury core based on the degree and extent of inflammation, crypt damage, and percent involvement17. Colitic mice showed signs of severe inflammation, while control mice receiving no DSS showed no histological damage (Figure 1D). The images demonstrate characteristic inflammatory changes in DSS-induced colitis, characterized by the destruction of epithelial architecture, with the loss of goblet cells, crypt damage, and mucosal ulceration, as opposed to the largely preserved mucosal architecture in control mice18. Statistical differences between the groups (n = 5 per group) were calculated by analysis of variance (ANOVA) followed by Student-Newman-Keuls (S.N.K.) post hoc test or by Welch's test followed by Games-Howell post hoc test in case of significant variance inhomogeneity. A p-value of < 0.05 was considered significant.
Assessment of Monocyte and Macrophage Recruitment:
As DSS-induced colitis is associated with the infiltration of immune cells, such as monocytes and macrophages, into the mucosa and submucosa of the colon, we used immunofluorescent staining for the monocyte/macrophage marker F4/80 to quantify the infiltrate. Colitic mice receiving DSS showed significantly elevated numbers of monocytes infiltrating the colon wall as compared to non-colitic control mice (Figure 2A). Leukocyte infiltration was additionally quantified using immunofluorescent staining for the neutrophil marker GR1, revealing the significantly increased immigration of neutrophils into the inflamed colon as compared to control mice (Figure 2B). To confirm, MPO levels reflecting both neutrophil number and inflammatory activity were significantly elevated in the colon tissue of colitic mice (Figure 2C). ANOVA and S.N.K. post hoc test were used to calculate the statistical significance between the groups (n = 5 per group). Statistical significance was set at p < 0.05.
Systemic Changes in Macrophage Subpopulation:
Mouse monocytes encompass a heterogeneous group of distinct subpopulations that differ in maturation state and their capacity to be recruited to inflammatory sites, where they participate in initiating and perpetuating the inflammatory response19. Therefore, we characterized blood monocytes by analyzing the differential expression of CD11b and Ly6C using flow cytometry. Under inflammatory conditions of acute DSS colitis, we observed a significant increase in inflammatory CD11bhighLy6Chigh monocytes as compared to pre-experiment conditions. In contrast, this shift did not occur in non-colitic control mice without DSS (Figure 3A). The data (n = 5 per group) were analyzed using ANOVA and S.N.K. As an additional surrogate parameter for systemic inflammation, we assessed the presence of F4/80-positive cells in the spleen of colitic mice compared to non-colitic controls, which revealed a significant increase in DSS-treated mice (Figure 3C).
Fluorescence-mediated Tomography Scan:
We used fluorescence-mediated tomography to measure monocyte/macrophage recruitment and infiltration into the gut mucosa. A fluorescence-labeled antibody against murine F4/80 was employed to directly visualize activated macrophages in living animals. Labeled, unspecific rat IgG antibodies were used as the isotype control. For scanning, mice were shaved in the abdominal region to minimize light reflection by the fur, anesthetized by continuous isoflurane supply, and examined in a veterinary FMT device for small-animal fluorescence. As shown in Figure 4A, the induction of colitis led to a significantly elevated accumulation of fluorescent tracer in the colons of colitic mice as compared to non-colitic controls, indicating an increase in monocyte infiltration and differentiation into active macrophages in colitic animals. The application of an unspecific fluorescence-labeled IgG did not elicit a detectable fluorescence response in the abdomen and intestine, neither in the colitic (DSS) nor the non colitic (water) group, demonstrating the specificity of the probe-target interaction of the F4/80 antibody (Figure 4A). Representative images of the abdominal region are shown. The color code indicates the level of fluorescence intensity, which corresponds to the extent of inflammatory infiltrate(Figure 4B and 4C). Ex vivo measurements of F4/80-directed tracer accumulation in explanted colons verified the colonic origin of the in vivo detected signal of F4/80 tracer accumulation (Figure 5A and 5B). Calculated amounts of bound antibody correlated well (R2 = 0.52) with histologically determined numbers of infiltrating macrophages (Figure 5C and 5D), confirming that in vivo imaging with specific tracers can be indicative of local disease activity. Data were analyzed by ANOVA and S.N.K. post hoc test with a statistical significance level set at p < 0.05. The correlation was calculated as linear regression.
Figure 1. Clinical parameters and histological injury over the course of acute DSS colitis.
C57BL/6 mice were challenged with DSS (2% w/v) in drinking water for 8 days. Control mice were given drinking water without DSS. Animals were euthanized on day 9. Shown are data from one experiment (n = 5 per group) ± the standard error of the mean (SEM). (A) Changes in bodyweight are presented relative to the initial weight. (B) Blood excretion in feces, as determined by the guaiac paper test (hemoccult, 0 = negative, 4 = blood macroscopically). (C) Post mortem colon length. (D) Histological injury as assessed by the degree of crypt damage and the extent of inflammation. Shown are representative histological images (haematoxylin/eosin staining; magnifications: 10x (upper panel), 20x (lower panel)) of colitic (left panels) or control (right panels) mice. Note the profound destruction of epithelial architecture and inflammatory infiltrates (arrowheads). Bar graph: injury score. Please click here to view a larger version of this figure.
Figure 2. Intestinal monocyte and macrophage infiltrate.
DSS colitis was induced in C57BL/6 by the application of DSS (2% w/v) in the drinking water. Control mice received drinking water alone. The data from n = 5 mice per group are shown ± SEM. (A) Immunofluorescent visualization of macrophage infiltrations in the mucosa. Shown are representative images of anti-F4/80 staining in colitic and control mice. Bar graph: cell count/high power field (HPF). (B) Immunofluorescent visualization of mucosa neutrophil infiltrations. Shown are representative images of anti-Gr-1 staining in colitic animals and controls. Bar graph: cell count/HPF. (C) MPO concentration in the colonic tissue of colitic and non-colitic mice. Please click here to view a larger version of this figure.
Figure 3. Monocyte recruitment to the intestinal compartment.
Peripheral blood monocytes from colitic mice as well as control mice (n = 5 per group) were analyzed by flow cytometry for the expression of Ly-6C on CD11b-positive cells. Cells were sorted based on SSC and CD11b expression. Mouse monocytes were identified as SSClowCD11bhigh cells, and their Ly6C expression was analyzed. (A) Proportional changes towards inflammatory Ly6Chigh monocytes are depicted relative to pre-experiment conditions (% change ± SEM). (B) FACS dot plots of Ly6C expression on SSClowCD11bhigh cells of colitic animals before and after colitis induction (lower panels) and controls pre- and post-experiment (upper panels). (C) Splenocytes from colitic and non-colitic mice were assessed for the expression of F4/80 by flow cytometry (mean fluorescence intensity ± SEM). Please click here to view a larger version of this figure.
Figure 4. Tomographic visualization of macrophage infiltrate in murine colitis.
FMT scans in colitic mice and non colitic controls administered Cyanine7-conjugated antibody against murine F4/80 (n = 5 per group). (A) Bar graph: total fluorescence intensity in pmol of dye was determined in the ROI and depicted ± SEM. Representative images are shown with color-coded fluorescence intensity corresponding to the extent of inflammatory infiltrate in mice injected with the specific probe (anti-mouse F4/80) (B) and an unspecific control (rat IgG) (C). In both cases, an ROI of equal size was placed transverse in the upper abdomen for further analysis. Please click here to view a larger version of this figure.
Figure 5. Ex vivo validation of tracer specificity in murine colitis.
In vivo FMT scans in colitic mice injected with the specific probe (anti-mouse F4/80) were followed by ex vivo planar fluorescence scans of explanted intestines to demonstrate the colonic origin of the tracer signal obtained in vivo. Data from two experiments with a total of n = 8 animals are shown. Representative images are shown depicting in vivo FMT scans of colitic mice with color-coded fluorescence intensity corresponding to the extent of inflammatory infiltrate (A). Fluorescence reflectance imaging of the explanted bowel allows for the identification of specific regions with tracer accumulation (B). Representative post mortem immunofluorescent staining for F4/80-positive macrophages from such areas confirms the in vivo results (C). The number of infiltrating macrophages, as determined by immunofluorescence staining, were correlated with in vivo measured tracer accumulation (D). Please click here to view a larger version of this figure.
Although medical imaging techniques have evolved rapidly in recent years, we are still limited in our ability to detect inflammatory processes or tumors, as well as other diseases, in their earliest stages of development. However, this is crucial to understanding tumor growth, invasion, or metastases development and cellular processes in the development of inflammatory disorders and degenerative, cardiovascular and immunological diseases. While traditional imaging techniques rely on physical or physiological parameters, molecular imaging enables the visualization of specific molecular markers in vivo20.
For small-animal imaging, radionuclide-driven approaches (e.g., single-photon emission computed tomography (SPECT) and positron emission tomography (PET)), ultrasound, and fluorescence-mediated imaging can be used to visualize specific molecular target structures. The use of full-length antibodies as the most available tracer backbone requires long-lasting labels, as long circulation times and slow tissue penetration delay the time of imaging after tracer application. Typical imaging radionuclides are therefore not suitable for the in vivo imaging of antibody distribution. The use of long-living isotopes like Cu64 or In111 enables antibody-based tracers but demands a complex infrastructure spanning isotope generation, radiation safety before and during the labeling procedure, and shielded animal housing after probe application. The more cost-effective and safer variant would be fluorescence-mediated imaging.
Several in vivo imaging systems for the detection, visualization, and measurement of fluorescent dye distribution in living animals have been presented over the past two decades21. Most systems enable rapid planar imaging suitable for superficial lesion imaging. Due to light absorption and scattering, signals deep in the tissue evade planar imaging. The most prominent solution for this problem is fluorescence tomography22. Based on mathematical modeling, the image signal is corrected for scattering and light absorption and a virtual 3D dataset is calculated from the multi-projection dataset8. The algorithm provides fully quantitative data, enabling the estimation of the tracer concentration in specific regions representative of the target concentration/expression23. A relevant limitation of FMT is related to poor spatial resolution as compared to anatomical imaging techniques, which might-depending on the desired target-compromise the allocation of molecular information to specific anatomical structures. Another limitation lies in the restricted penetration depth of light into tissue, which requires the use of probes absorbing and fluorescing in the red to NIR spectral range23.
A recently introduced imaging modality that combines the advantages of morphological ultrasound and the possibility of obtaining molecular information from fluorescence imaging is optoacoustic imaging. Preliminary results from early studies suggest that-despite a need for further technical refinement-optoacoustic imaging holds great promise for molecular imaging and potential translational applications24.
The recruitment of leukocytes to the intestinal wall is a crucial step in the initiation and perpetuation of IBD, as is the recruitment of inflammatory cells to the site of infection or inflammation in many immune-mediated disease, such as autoimmune disease, infection, vascular disease, tumorigenesis, and many more25. As monocyte activation and infiltration into intestinal mucosa is an essential step in the pathogenesis of IBD, inhibition of monocyte infiltration, orchestrated by chemokines and integrin-cellular adhesion molecule interaction, is a promising therapeutic approach12,26. Therefore, monitoring leukocyte trafficking to the site of inflammation is crucial in the development of new therapeutic options to visualize the anti-inflammatory effects of the studied drugs. This may be of special interest because agents targeting leukocyte trafficking have been developed, and some anti-integrin antibodies are already available for IBD therapy, while further components will be available in the near future26. Vedolizumab, a humanized monoclonal antibody against the gut-specific α4β7 integrin subunit, and Etrolizumab, which binds the β7 subunit of the intestinal α4β7 and αEβ7 integrin heterodimers, have been approved27,28. Other agents (e.g., the MAdCAM-1 PF-00547659 and sphingosine-1-phosphate (S1P) receptor modulators such as Ozanimod) are currently undergoing evaluation for the treatment of IBD29,30.
When performing FMT, possible modifications of the technique include the selection of different tracer-target combinations, as well as combining FMT with structural imaging approaches to compensate for poor spatial resolution. A wide array of probe-target combinations has been established and commercialized. For specific projects, the labeling of custom antibodies can be done using commercial labeling kits and the above-described protocol. Depending on the antibody or smaller peptide chosen as the targeting moiety, commercial suppliers offer a wide array of different labels. Consider the desired dye, depending on the application: for deep-tissue imaging, a dye operating in the NIR spectrum (e.g., Cy7, λex/em 750/780 nm) might be well-suited, while the overall brightness and effective light supply of dyes operating in the near-visible range of the spectrum (e.g., GFP analogues) might be preferable. Virtually all dyes can be obtained as an active ester to label the lysine- residues of proteins, as well as with alternative binding moieties, such as maleimides for cysteine binding or biotin for the labeling of proteins equipped with specific labeling tags31. The selection of the label does not change the principle protocol but merely requires slight modifications of the labeling procedure and is important for a good imaging result. Another attractive modification to overcome the limitations in spatial resolution is to combine FMT with CT or MRI, enabling the annotation of anatomical structures with molecular information. The structural information can either be acquired sequentially as a priori information or simultaneously, which requires hybrid imaging instrumentation32,33.
Certain steps of the protocol deserve special attention. As this technique can be adapted for a multitude of fluorescent photoprobes that target a variety of specific molecules representative of different molecular processes, it is essential to allow for the sufficient distribution of the probes and enrichment in the desired target region. The ideal time point for imaging might vary according to the probe-target combination. For example, antibody targeting of tumor receptors might be influenced by the diffusion rate to tumors, uptake and metabolism by the liver, and possible adverse immunogenic reactions34. Also consider relevant controls for specific imaging approaches. Specific tracers should always be validated against isotype controls reflecting perfusion effects and unspecific binding (e.g., Fc receptor-mediated binding). Target-negative animals can serve as an additional control for tracer specificity, if such knockout models exist. Alternatively, blocking experiments can be performed to prove the specificity of antibody-target interactions35.
The following are critical steps concerning the practicalities of the procedure: (1) Carefully determine the DSS concentrations used, as DSS susceptibility might vary between different mouse strains and manufactures/batches36. (2) Carefully select probe-target combinations. (3) Allow around 5 min of scan time for a sufficient scan of the whole abdomen. The optimal time point between tracer application and the imaging procedure needs to be carefully determined to allow for tracer accumulation in the desired target region. For intestinal F4/80 detection in murine colitis, the labeled antibody should be injected 24 h prior to scanning. (4) As unspecific label accumulation might occur (e.g., in the liver), it is crucial to label the appropriate target tissue as ROI by placing the respective measuring tools. Moreover, sufficient controls for unspecific tracer distribution should be included-consider unspecific isotype controls to rule out perfusion effects and knockout or blocking studies to validate tracer-target interaction.
Typical sources of error related to the scanning procedure and data reconstruction include improper animal position, scan field, and exposure time. When positioning the animal, the fluorescing lesion should be surrounded by tissue on all sides to minimize reconstruction noise. Air bubbles trapped between the animal and the front glass plate of the imaging cassette might also increase noise and should be removed by moving the animal slightly. Over- or underexposure of the image can require a reacquisition scan with modified exposure settings, which can be found in the scan screen (reflectance image block: adjustments of the front LED intensity and exposure time). When combining in vivo antibody-based FMT measurements with post mortem histological analyses, such as immunohistochemistry or immunofluorescent staining, the use of antibodies of the same clone and format should be considered. Also, when performing repetitive FMT measurements in the same animals, the same formats and clones should be used to prevent possible cross-reactivity or the targeting of different epitopes.
Taken together, fluorescence mediated-molecular tomography enables the repetitive monitoring of the disease course and underlying pathophysiological processes. It can be applied to a plethora of biomedical studies and might be useful to accelerate drug testing or as an objective endpoint in individualized therapies. FMT-targeting of F4/80-expressing macrophages in models of inflammation provides a valuable noninvasive tool to visualize and quantify the infiltration and accumulation of inflammatory cells while also demonstrating good correlation to conventional readouts.
The authors have nothing to disclose.
We thank Ms. Sonja Dufentester, Ms. Elke Weber, and Mrs. Klaudia Niepagenkämper for the excellent technical assistance.
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 | |
Name | Company | Catalog Number | Comments |
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 |