A P P L I C AT I O N N O T E

VivoTrack 680
AuthorJeffrey D. Peterson, Ph.D.

PerkinElmer, Inc.
940 Winter Street
Waltham, MA USA

A method of NIR
fluorescent celllabeling for in vivo
cell tracking

Abstract

Fluorescent dyes have been used for many years
to label cells for microscopy studies, and a varietyof dyes in the visible fluorescence spectrum areavailable to label different cellular compartments
and organelles. Efficient delivery of the fluorophore
to the cell without excessively modifying surface
proteins or perturbing cell function is the major
biotechnological challenge. In addition, researchers
have taken on the challenge of in vivo imaging, focusing on near infrared (NIR) dyesthat fluoresce in a spectral region better suited for in vivo imaging due to reduced
background and higher tissue penetration. Here we introduce VivoTrack™ 680, a novel
NIR dye that has a long aliphatic tail to facilitate intercalation into the lipid bilayer ofthe cell. This agent can label a variety of cells brightly and uniformly without requiringharsh organics such as DMSO or ethanol, chemicals known to affect cell function. Thelabeled cells retain excellent viability and normal proliferative capacity, and significant
signal is retained in dividing cells in vitro for more than 7 days. Many different cell
types can be labeled for in vivo transfer or implantation, and in vivo utility is illustrated
by non-invasively tracking fluorescent primary macrophages to sites of inflammation
by NIR fluorescent imaging.

MATERIALS AND METHODS:
Fluorescent Cell Labeling Dye

VivoTrack Labeling of Cells

VivoTrack 680 is a near infrared (NIR) dye with an long aliphatic
tail that facilitates integration into the cell membrane for thepurpose of identifying cells in vitro or tracking them in vivo.
This agent provides a simple and rapid method for labeling cells
without affecting cell viability, proliferation, and function.

1. Wash the cells of interest once with sterile PBS or serum-free
medium to remove serum proteins and lipids that may interferewith cell labeling.

Table 1.
Basic Properties of VivoTrack 680 Fluorescent Cell Labeling Agent
VivoTrack 680
Agent Type

Lipophilic dye

Molecular Weight

1173 g mol-1Absorbance

676 nmFluorescence emission

696 nmCells and Cell Lines
The mouse leukemic monocyte macrophage cell line RAW
264.7, was obtained from American Type Culture Collection(ATCC, Manassas, VA). Cells were routinely cultured in RPMI
medium supplemented with 10% fetal bovine serum and 1%
penicillin-streptomycin in 75 cm2 flasks. Exponentially growing
cells between passages 1-10 were used for all experiments.
Cells were labeled with VivoTrack 680, as described below
and analyzed by flow cytometry (Becton Dickinson LSRII)
and fluorescence microscopy (Zeiss Axioskop 2 MOT Plus,Oberkochen, Germany) using appropriate lasers and filters for
detection of 680 nm wavelength. Cell nuclei were stained withDAPI (blue).
Mouse thioglycollate elicited peritoneal macrophages werealso used as a source of primary cells for labeling and transferstudies. Briefly, a thioglycollate solution was prepared byboiling 3% Brewer thioglycollate medium in distilled water
followed by storage at room temperature. Normal BALB/cmice were injected intraperitioneally with 1.5 mL ofthioglycollate medium per 25 g body weight. This injection
induced a proinflammatory response, generally yielding~1 x 107 macrophages per mouse 3 days later. These cells wereharvested from sacrificed animals by peritoneal lavage using 3
separate washes of the peritoneum for a total of 10 mL PBS.
Subsequently, cells were washed with 1 X PBS and counted forlabeling. Red blood cells were lysed as necessary.

2. Discard the supernatant and resuspend the cells (up to 250 x106 cells/mL) in 2.0 mL of PBS in a 50 mL sterile conical tube.
3. Dissolve VivoTrack 680 (1 g) in 1.2 mL of warm sterile PBS(37 °C) and mix by vortex until completely dissolved. This will
yield 2.0 mL of the labeling agent.
4. Add 2.0 mL of the cell labeling solution to 2.0 mL of cells, andmix immediately by gentle vortexing. [Note: It may be required
to optimize dose depending on the cells to be labeled.]
5. Incubate the cells for 15 min at room temperature, protectedfrom light.
6. Dilute the cells for washing by adding 15-20 mL sterile RT PBS
containing 1% FBS or complete medium, depending on yourultimate use for the cells.
7. Wash the cells 3 times with RT PBS containing 1% FBS toremove excess cell labeling agent. A final resuspension withsterile PBS alone can be used to decrease the FBS levels in thecell preparation.
8. Count, culture, or transfer cells as required by the application.
VivoTrack 680 is highly soluble in aqueous solution and,thus, does not need to transition from organic to aqueous
solution upon addition to the cell suspension. This allows quick
dispersion into the cell medium, and very uniform cell labelingis generally achieved.
In Vivo Macrophage TrackingMouse carrageenan-induced paw edema, a model of acuteneutrophil-mediated inflammation (Figure 1), was used for thepurpose of recruiting macrophages to inflamed paw tissue. Briefly,
BALB/c mice received an intravenous injection of 5 x 106 VivoTrack
680-labeled thioglycollate-elicited peritoneal macrophages toprovide a reservoir of fluorescent-labeled cells available forrecruitment in response to the carrageenan stimulus. Twentyfour hours later, each mouse received a 30 mL injection of PBS(left paw) and 1% carrageenan (right paw). Some mice weredepilated completely to allow fluorescent imaging of whole body
biodistribution of labeled
macrophages at 24 h.
Optimal macrophage
recruitment occurred
within 3 h of carrageenan
injection (i.e. 27 hfollowing macrophageinjection) and this was
easily detected andquantified in paws of
un-depilated mice.
Figure 1.

2

Imaging Data Analysis

VivoTrack 680 In Vitro Characterization

Epifluorescence (2D) images were captured at 3 h post-carrageenan
injection by the IVIS® Spectrum CT imaging system (PerkinElmer,Waltham, MA). The FMT 4000 (Fluorescence MolecularTomography) imaging system (PerkinElmer, Waltham, MA) wasused to capture both epifluorescence and tomography [3D]
datasets. To enable transillumination of the paws for tomographic
imaging, paws were placed over an index-matching block.

Table 1 provides the physical-chemical characteristics and theabsorbance and emission maxima, and Figure 2 shows the full
spectral profile of VivoTrack 680. Cells are labeled brightly, withgood cell-to-cell consistency, as shown by labeling of both amonocyte/ macrophage cell line (RAW 264.7), as well as primary
thioglycollate-induced peritoneal macrophages (Figure 3).

Imaging datasets for the IVIS Spectrum were visualized andquantified by Living Image® 4.3.1 software, and FMT data wasanalyzed by FMT system software (TrueQuant™ v4.0), bothfrom PerkinElmer (Waltham, MA). Regions of interest (ROI),
2D and 3D as necessary, were drawn encompassing each paw,
with the PBS injected paws serving as the negative controls.

Introduction and Results
Understanding the pathophysiology of disease processes atthe cellular and biochemical level is a major challenge in basic
research and drug development. The complexity of theseprocesses, and the difficulty of recapitulating this complexity
in vitro, raises the importance of robust and quantitative invivo techniques to study specific cellular players in diseaseprogression. In inflammation, for example, the ability to observeand quantify the recruitment of leukocytes from the blood tosites of tissue infection or trauma is crucial for understanding theinduction of acute or chronic inflammatory tissue destruction.
Fluorescent dyes have been invaluable tools for this type of cell
monitoring in vitro and in vivo, but today there remains the needfor fluorescent labeling agents that can provide 1) uniform celllabeling, 2) long cellular residence time, 3) minimal impact oncell viability and function, and 4) a near infrared wavelength for
optimal light penetration of tissue.
The VivoTrack 680 cell labeling agent provides a simple andeasy NIR membrane labeling tool that is specially formulated
for excellent solubility in the absence of organic solvents likeethanol or DMSO. This agent intercalates into cell membranes(primary macrophages and RAW264.7 cells here), providing abright label by which cells can be tracked in vivo. A panel ofstudies determined that cells were consistently and brightly
labeled as assessed by both flow cytometry and fluorescent
microscopy, and cells retained excellent viability andproliferation. Primary mouse macrophages were readily
tracked to a site of acute inflammation.

Figure 3.
RAW 264.7 cells and peritoneal
macrophages (thioglycollateelicited) were labeled 15 minuteswith 20 μM VivoTrack 680 (red)and DAPI nuclear stain (blue).
DAPI only cells were used toshow the lack of backgroundsignal in the absence of labeling.

The brightness of cell labeling by VivoTrack 680 was determinedby flow cytometry using the LSR II system (Becton Dickinson,Franklin Lakes, NJ) modified to include a 660 nm laser for
NIR imaging. Cell proliferation/viability was assessed by two
independent methods; WST assay and BrdU incorporation kits
(Roche Diagnostics GmbH, Mannheim, Germany). Figure 4
shows that labeling of RAW264.7 cells with VivoTrack 680 yields
bright cells with no effect on viability as assessed by both WSTand BrdU incorporation assays.

Figure 4.

Figure 2.

RAW 264.7 cells were assessed for brightness and viability after 15 minute incubations with either vehicle or VivoTrack 680. Flow cytometry was used to assess
fluorescence, and microplate-based assays were used to quantify viability.

VivoTrack 680 absorption
and emission spectra
show peak absorption
at 676 nm and peakemission at 696 nm.
3Rapidly dividing cells are expected to lose membrane fluorescentsignal over time, however signal is readily detectable for at least
7 days when labeling RAW264.7 cells with VivoTrack 680 (Figure5). Fluorescence retention kinetics are related to the rate of cell
division, and, thus, may depend on the type of cell labeled.

Figure 5.
The effect of RAW 264.7 cell proliferation on fluorescence levels after VivoTrack 680
labeling was assessed by maintaining cells in culture for up to seven days. Cells wereassayed on days 0, 3, and 7, revealing a consistent rate of loss proportional to theoverall increase in cell numbers.

VivoTrack 680 In Vivo Characterization
To assess the in vivo performance of VivoTrack 680 labeled
macrophages, we used a simple mouse model of acute neutrophildriven inflammation, carrageenan-induced paw edema. Briefly,thioglycollate-elicited macrophages from donor BALB/c mice werelabeled with VivoTrack 680 and then transferred intravenously (5 x106 cells/mouse) into normal BALB/c mice. The labeled cells wereallowed to accumulate in the liver and lungs for 24 h, and thenthe recipient mice were injected with carrageenan (right footpad)
and PBS (left footpad). The active site of inflammation was used asa means to recruit inflammatory cells, including the VivoTrack 680
labeled macrophages injected 24 h earlier.
We used non-invasive fluorescence imaging and IVIS Spectrum
imaging systems to assess these changes three hours after
carrageenan injection (Figure 6). Macrophages selectively trafficked
into inflamed paws and accumulated there, showing statisticallysignificant elevations in fluorescence. As expected, the peak
macrophage influx (3 – 24 h) accounted for ~0.4-2% of the total
injected cells (20-100,000 cells/mouse).
The kinetics of labeled macrophage accumulation in inflamed paws

Figure 6. BALB/c mice received an intravenous injection of labeled thioglycollate-elicited peritoneal macrophages to provide a reservoir of fluorescent-labeled cells

available for recruitment in response to an inflammatory stimulus. One cohort of mice was depilated completely to allow FMT 4000 tomographic imaging of whole body
biodistribution of labeled macrophages at the time of carrageenan injection (A) for comparison to tomographic paw imaging 3h after carrageenan (CG) injection (B). FMT
quantification shows that the paw signal is approximately 0.4% of the total body signal, indicating that ~20,000 cells were recruited to the inflamed paw (C). Epifluorescenceassessed by IVIS (D) and FMT (E) each offer a rapid screening approach for the measurement of fluorescent macrophage recruitment to the inflamed paws (F), but not tothe control, PBS-injected paws. Quantification of fluorescence datasets was performed with some modest thresholding to remove non-specific background fluorescence.
4

was assessed by imaging mice at multiple times after CG injection(Figure 7). Changes in paw thickness over time were also assessedby engineer’s micrometer, indicating a peak in inflammation at 3 hwhich decreased steadily by 72 h (Figure 7A). A lower level chronic
phase of paw swelling was observed between 72 and168 h, perhaps reflecting a transition from acute neutrophilia
to chronic host macrophage accumulation. The kinetics of therecruitment of injected fluorescent macrophages, measured by
epifluorescence imaging, also peaked at 3 h and declined steadily
until 168 h (Figure 7B), aligning well with peak inflammation andthe likely secretion of cytokines and chemokines. Signal loss wasattributed to macrophage efflux and/or loss of fluorescence due
to in situ proliferation or death. This model would offer an ideal
approach to assessing drugs or antibodies that affect chemokinesor adhesion molecules involved in macrophage recruitment.

Summary
PerkinElmer has developed a near infrared fluorescent celllabeling agent, VivoTrack 680, that can generate brightly-labeled
and highly viable cells suitable for detection and longitudinal
tracking in vivo. As cell migration is critical for many importantin vivo biological functions, this labeling approach has thepotential to provide a useful tool for research in inflammation,
immunology, and stem cells.

Figure 7.
A representative BALB/c mouse recipient of VivoTrack 680-labeled macrophages
was imaged by FMT epifluorescence at multiple times post transfer (A).
Quantification of paw swelling and macrophage recruitment (B) were assessed byengineer’s micrometer measurement of paw thickness and quantification of paw
epifluorescence, respectively. Epifluorescence data was thresholded to minimize
non-specific background signal in negative control paws to below 20 counts/energy.

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March 2013