Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Podsumowanie

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Streszczenie

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Wprowadzenie

Liposomes have been intensively investigated and serve as one of the most biocompatible biomedical drug delivery systems for clinical applications^1,2. They are mainly composed of phospholipids and cholesterol, both of which are biocompatible compounds mimicking parts of natural cell membranes. Whereas hydrophilic substances can be entrapped in the aqueous interior, lipophilic agents can be incorporated within the liposomal phospholipid bilayer³. Encapsulation of substances within the aqueous interior of liposomes grants protection against degradation in vivo and also prevents the host system from toxic effects of cytotoxic drugs used for the therapy of diseases, for example chemotherapeutics aimed at destroying tumor cells. The modification of the liposomal surface with polymers like polyethylenglycol (PEGylation) further extends the liposomal blood circulation time in vivo due to sterical stabilization⁴. Moreover, liposomes can sequester high concentrations of several substances such as proteins^5,6, hydrophilic substances^7,8 and enzymes⁹. They therefore serve as reliable clinical therapeutic and diagnostic tools which merit their approval for delivery of cytotoxic drugs such as doxorubicin for cancer therapy⁴. Due to their flexibility, liposomes can also be loaded with fluorochromes for diagnostic and image-guided surgical purposes.

Fluorescence imaging provides a cost-effective and non-invasive in vivo diagnostic tool which however, demands some basic requirements. It could be demonstrated that fluorochromes which suit best for in vivo imaging have characteristic absorption and emission maxima in the range where light dispersion and scattering as well as tissue autofluorescence originating from water and hemoglobin is low. Thus, such probes have their abs/em maxima between 650 and 900 nm¹⁰. Besides this, the stability of fluorochromes both in vitro and in vivo is critical, as opsonization and rapid clearance can greatly limit their application for in vivo imaging¹¹. Other effects such as poor stability and low sensitivity or cytotoxic effects on target organs as seen with indocyanine green (ICG)^12-16, are unwanted and must be taken into consideration when designing probes for in vivo imaging. These observations have led to the active development of several preclinical NIR fluorochromes, nanoparticles as well as new techniques for the in vivo imaging of inflammatory processes, cancer and for image-guided surgery^17-20. Despite the stability of most preclinical NIRF (near-infrared fluorescence) dyes in vitro, their rapid perfusion and clearance through the liver and kidney impede their use in the in vivo optical imaging of diseases and inflammatory processes.

We therefore present a protocol for the encapsulation of fluorochromes such as the well characterized near-infrared fluorescent dye DY-676-COOH, known for its tendency to self-quench at relatively high concentrations²¹ in liposomes. At high concentrations H-dimer formation and/or pi-stacking interactions between fluorophore molecules located within each other’s Förster radius result in Förster resonance energy transfer (FRET) between the fluorochrome molecules. At low concentration the space between the fluorophore molecules increases, thereby preventing pi-stacking interaction and H-dimer formation and resulting in high fluorescence emission. The switch between high and low concentration and the accompanying fluorescence quenching and activation is a promising strategy that can be exploited for optical imaging²². In this respect, encapsulation of high concentrations of the NIRF dye DY-676-COOH in the aqueous interior of liposomes is more favorable for in vivo imaging than the free dye. The challenge of the method lies first of all in the correct encapsulation and secondly, in the validation of the benefits resulting from encapsulating high concentrations of the dye. Comparing the imaging properties of quenched liposomes with that of the free dye and also with a non-quenched liposome formulation with low concentrations of the dye is indispensable. We show by a simple, but highly effective film hydration and extrusion protocol combined with alternate freeze and thaw cycles that encapsulation of quenching concentrations of DY-676-COOH in liposomes is feasible. Other methods used to prepare liposomes such as the reversed phase evaporation method²³ as well as the ethanol injection method²⁴ enable liposome preparation with high encapsulation efficiencies for many hydrophilic substances. However, the nature of the substance to be encapsulated can influence the encapsulation efficiency. In effect, the film hydration and extrusion protocol presented here revealed the highest efficiency for encapsulation of DY-676-COOH. To illustrate the benefits of liposomal encapsulation of DY-676-COOH, a zymosan-induced edema model, which permits the study of inflammatory processes within a few hours, was used. Here, it is demonstrated that liposomes with high concentrations of the encapsulated DY-676-COOH are more suitable for whole body in vivo optical imaging of inflammatory processes than the free dye or the non-quenched liposomal formulation with low dye concentrations. Thus the underlying protocol provides a simple and fast method to produce quenched fluorescent liposomes and the validation of their activation and imaging potential both in vitro and in vivo.

Protokół

NOTE: All procedures are approved by the regional animal committee and in accordance with international guidelines on the ethical use of animals.

1. Preparation of Materials and Instruments

1. Preparation of spontaneously formed vesicle dispersion (SFV)
   1. Dissolve and prepare stock solutions of the following phospholipids: 214 mg/ml egg phosphatidylcholine (EPC), 134 mg/ml cholesterol, 122 mg/ml 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (mPEG₂₀₀₀-DSPE) and 2 mg/ml 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD-DOPE) in chloroform and store in glass vials.
   2. Furnish approximately 3 ml chloroform in a round bottom flask and transfer the appropriate volume of phospholipid stock solution into the round bottom flask to prepare liposomes composed of EPC:Chol:mPEG₂₀₀₀-DSPE at a molar ratio of 6.5:3:0.5. For double fluorescence labeling of liposomes add 0.3 mol% NBD-DOPE to the lipid solution.
   3. Evaporate the chloroform from the organic phospholipid solution under reduced pressure (300 mbar) at 55 °C using a rotary evaporator.
   4. After a homogeneous phospholipid film is formed, reduce the pressure to 10 mbar for 1-2 hr to remove residual chloroform deposit.
   5. While the chloroform is evaporating, dissolve DY-676-COOH (6.181 µM) in 10 mM Tris buffer pH 7.4 and fill a Dewar vessel with liquid nitrogen. Switch on an ultrasonic bath and set at 50 °C.
   6. Transfer an appropriate volume (0.5–1 ml) of DY-676-COOH (6.181 µM) solution to the round bottom flask to hydrate the dry phospholipid film and vortex vigorously until a spontaneously formed vesicle (SFV) dispersion forms. Make sure that all of the phospholipids are dispersed to avoid lipid loss.
   7. Carefully transfer the round bottom flask containing the SFV dispersion into liquid nitrogen and freeze the dispersion for 3–5 min. Place the round bottom flask into an ultrasonic bath at 50 °C to thaw the dispersion then vortex the dispersion vigorously for 1–2 min. Repeat this procedure six times, making a total of seven freeze and thaw cycles.
2. Extrusion of SFV to form homogeneous liposome vesicles
   1. Transfer the SFV dispersion into a 1 ml syringe (syringe-a) and extrude the dispersion through a 100 nm polycarbonate membrane using a LiposoFast-Basic extruder into syringe-b.
   2. Extrude the dispersion from syringe-b, back into syringe-a, then repeat the cycle ten times. Due to extrusion, the solution in the syringe changes from a hazy appearance to a clear dispersion with time. After ten cycles (twenty single extrusion steps) remove syringe-b from the device and extrude the dispersion for the last time from syringe-a directly into a sterile 1.5 ml reaction tube.
3. Purification of liposomal encapsulated DY-676-COOH from free dye
   1. Prepare a gel chromatography column using G25 beads soaked in 10 mM Tris buffer pH 7.4 (column length 28 cm, diameter 0.8 cm).
   2. Transfer 0.5 ml of extruded vesicle dispersion onto the gel bed and let the sample drain into the gel matrix.
   3. Elute the liposomes with 10 mM Tris buffer pH 7.4 (Figure 1A) and wash the column until the free dye drains completely out of the column. If need be, collect and recycle the free dye by desalting and dehydration according to the manufacturer’s instructions.
   4. Concentrate the eluted liposomes by ultracentrifugation (200,000 x g, 2 hr at 8 °C) then disperse them in adequate volume of sterile 10 mM Tris buffer pH, 7.4.
4. Quantification of encapsulated DY-676-COOH concentration
   1. Prepare a calibration curve by dissolving DY-676-COOH (0, 82, 124, 247, 494, 988 nM) in 10 mM Tris buffer, pH 7.4 containing 0.1% Triton X100.
   2. Dissolve 2 µl (100 nmol final lipid of a 50 mmol/L stock) of the liposomes for 5 min at room temperature in 100 µl Tris buffer containing 1% Triton X100 to destroy the vesicles and release the encapsulated dye. Then dilute samples with 10 mM Tris buffer, pH 7.4 to a final Triton-X100 concentration of 0.1% (v/v) making a total volume of 1 ml. Prepare all samples in duplicate.
   3. Measure the absorption and emission of all samples (free DY-676-COOH and Triton-X100 treated liposomes) at an excitation λ=645 nm and an emission λ = 700 nm. Establish and use a calibration curve of the free dye to determine the concentration of encapsulated dye.
5. Liposome characterization
   1. Determine the sizes and zeta potential of liposomes by dynamic light scattering. Dilute the liposomal samples with filter sterilized (0.2 µm) 10 mM Tris buffer pH 7.4 to a concentration of 100–300 µM (lipid). Transfer the diluted samples into a low volume disposable cuvette and measure the sample according to the manufacturer’s instructions.
   2. Characterize the liposomes by electron microscopy to substantiate the size, integrity and homogeneity of liposomal vesicles according to standard protocols.

2. Validation of Fluorescence-quenching and Activation of Prepared Liposomes

1. Physicochemical analysis of fluorescence quenching and activation
   1. Prepare two 1.5 ml tubes for the Lip-Q and 2 tubes for the free DY-676-COOH. Transfer 100 nmol total lipids (2.38 µl of a 42 mmol/L Lip-Q stock solution, containing 138 µg/ml of the encapsulated DY-676-COOH) into the corresponding tubes. Transfer free DY-676-COOH equivalent to the dye content of Lip-Q (0.38 µg which results for example from 138 µg/1,000 µl x 2.38 µl Lip-Q used). Incubate one tube of each probe at 4 °C and freeze the second tube at -80 °C overnight (16 hr).
   2. Heat up a heating block to 30 °C. Fill a cooling box with crushed ice and equilibrate an aliquot of 10 mM Tris buffer pH 7.4 to room temperature.
   3. Remove probes from 4 °C and equilibrate at room temperature (wrapped in aluminum foil to protect from light) and quickly thaw probes from -80 °C at 30 °C for 5 min. Chill the thawed probes on ice for 1 min before transferring them to room temperature (also wrapped in aluminum foil to protect from light).
   4. Add 10 mM Tris buffer (pH 7.4) to each of the probes to 100 µl final volume and equilibrate all probes at RT for 10 min.
   5. Pipette 80 µl of each probe into a low volume glass cuvette and measure the absorption of each probe from 400–900 nm on a spectrometer. Return the probe to their corresponding tubes.
   6. Transfer 80 µl of each probe into a suitable glass cuvette and measure the fluorescence emission on a spectrofluorometer by exciting the probes at 674 nm and measuring the fluorescence from 694–800 nm.
2. Cellular uptake and fluorescence activation
   1. Get and culture the following cell lines in their corresponding culture media according to standard conditions (37 °C, 5% CO₂ and 95% humidified atmosphere). Here, use the murine macrophage cell line J774A.1 (Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum), the human glioblastoma cell line, U-118MG (MEM containing essential vitamins and 10% (v/v) fetal calf serum) and the human fibrosarcoma cell line, HT-1080 (RPMI with 5% FCS).
   2. Coat 8-well chamber slides with poly-L-lysine (add 100 µl 0.001% poly-L-lysine to each well and incubate at 37 °C for 10 min. Aspirate solution and let the chamber slides to dry up for at least 4 hr at RT on a sterile workbench. Rinse the chamber slides 3 times with 200 µl Hank’s buffered saline solution then seal them with paraffin and aluminum foil and store at 4 °C till needed.
   3. While the chamber slides are drying up, filter-sterilize the liposomes and dye solution and store at 4 °C till needed.
   4. For probe uptake analysis with the whole body in vivo NIR fluorescence imaging system, prepare 5 small culture flasks for each of the 3 test cell lines (15 flasks in total). Seed 2 x 10⁶ J774A.1, U-118MG and HT-1080 cells per culture flask with 5 ml of the respective culture medium (in quintuples) and grow for 16-24 hr. Parallel to the culture flasks, seed 30,000 cells of each cell line (J774A.1 and HT-1080) or 20,000 cells (U-118MG) to 2 wells of the chamber slide respectively and grow in 500 µl culture medium for 16-24 hr.
   5. On the next day, add 100 nmol (final lipid amount) of Lip-Q to 2 flasks per cell line and to one well of each cell line on the chamber slide. Immediately transfer 1 flask per cell line to 4 °C and the second flask back to the incubator.
      NOTE: The volume of the probes added to the flasks and the chamber slides are the same, making the concentration on chamber slides 10-fold higher (5 ml is to 500 µl culture medium). This is necessary because microscopic detection is less sensitive than the NIR fluorescence imaging system where cell pellets from the flasks are imaged.
   6. Add the free DY-676-COOH at a concentration equivalent to the dye content of Lip-Q to the cells in 2 flasks per cell line and to one well of each cell line on the chamber slide, then immediately transfer 1 flask per cell line to 4 °C (energy depletion) and the other flask together with the chamber slide back to the incubator. Incubate all the cells for 24 hr at the corresponding conditions. The cells in the flask without probe serve as untreated control.
3. NIRF imaging and semi-quantitative analysis
   1. After 24 hr incubation duration, harvest the cells in flasks by washing cells 2 times with Hanks buffered salt solution (HBSS) then scrape cells in 500 µl HBSS and pellet by centrifugation (5 min at 200 x g) in 500 µl tubes.
   2. Place the tubes with the cell pellets (and HBSS) in a NIRF imager and image using filters for excitation (615–665 nm) and emission (cut in >700 nm).
   3. Deduct autofluorescence and evaluate the intensity of target versus autofluorescence according to manufacturer instructions. This will give the semi-quantitative levels of fluorescence intensity as average signal (scaled counts/sec), which represents count levels after scaling for exposure time, camera gain, binning and bit depth, so that the measurements are comparable among each other.
4. Confocal microscopic analysis
   1. After 24 hr incubation, harvest the cells on chamber slides by washing 2 times with 500 µl HBSS.
   2. Fix the cells with 200 µl HBSS containing 3.7% (v/v) formaldehyde for 30 min at RT.
   3. While fixation is going on, dilute the DNA stain, Hoechst-33258 1:50 with mounting solution.
   4. After fixation, wash the cells 2 times with HBSS then separate the chambers from the glass slides. Add 50 µl mounting solution containing the DNA-stain on each spot corresponding to the wells of the chamber slides. Cover the cells with glass coverslips, seal edges with transparent nail polish and air-dry for 10 min at RT (dark).
   5. Image cells on a suitable fluorescence microscope or confocal microscope. Use the following excitation and emission settings for visualization of the corresponding components: nuclei (Hoechst-33258: excitation 405 nm, emission 420–480 nm). NBD-DOPE (liposomal lipid: excitation 488 nm, emission 530 nm). DY-676-COOH (NIR fluorescent dye: excitation 633–645 nm, emission 650–700 nm).
      NOTE: Make sure that the fluorescence microscope available is equipped with suitable filters which permit excitation and emission of wavelengths higher than 630 nm.

3. Liposome-based In Vivo Fluorescence Imaging of Inflammation

1. Preparation of animals and materials
   1. House 8–12 week-old male NMRI mice weighing approximately 36 g under standard conditions with food and water ad libitum.
   2. Seven days prior to the start of experiments, give all mice a low pheophorbide diet in order to reduce tissue autofluorescence.
   3. Twenty-four hours before the start of each experiment, shave mice at the desired area (e.g., whole back area if imaging of hind leg edema is desired).
   4. Weigh the animals and calculate the amount of probe to be injected per mouse (Lip-Q and Lip-dQ at 10 µmol (lipid concentration) per kg weight and free DY-676-COOH (equivalent to dye content of Lip-Q used).
   5. Image the animals in a whole body NIR Fluorescence imager with the same settings used for the cell pellets. This measurement provides autofluorescence of the animals.
   6. Dissolve 10 mg zymosan-A in 1 ml isotonic saline solution and store overnight at 4 °C.
2. Induction of inflammation and in vivo NIRF imaging
   1. Prepare 3 syringes per mouse containing the following solutions. Fill one syringe with 50 µl zymosan-A solution (10 mg/ml) and the second syringe with 50 µl isotonic saline solution. Fill the third syringe with the probes, whereby Lip-Q and Lip-dQ (10 µmol/kg weight (lipid)) are designated for test animals and free DY-676-COOH (concentration as in Lip-Q) for control animals. Make sure the probes are diluted with sterile HBSS to 150 µl final volume.
   2. Apply eye cream on the eyes of animals to avoid dryness and anesthetize animals with 2% isoflurane till they are deeply asleep and do not react when touched on the paws (this takes about 2 min).
   3. Place the mouse on a warm mat (still under anesthesia) and inject the zymosan-A solution subcutaneously on the right hind leg and the saline solution on the left hind leg. Immediately inject the probe intravenously and image the animal thereafter then record time of injection/measurement (as t = 0 hr). Save the resulting images as image cubes and repeat the above steps for all other animals and respective probes.
   4. Image the animals every 2 hr for 10 hr post injection and then at 24 hr post injection, making sure that the stage of the measuring chamber is warm (for example, by placing a warm mat underneath), in order to avoid hypothermia. After each measurement, place the animals in a cage with food and water ad libitum and place the cage in a temperate animal chamber.  Euthanize the animals by first anesthetizing with 2% isoflurane till the animals no longer react to touch, then euthanize with carbon dioxide for 5-10 min, making sure that the animals stop breathing completely and rigor mortis occurs. 
   5. Dissect the mice according to standard protocols which can be assessed online (http://www.freebookez.com/mouse-dissection-lab-report/) and image the organs.
   6. Evaluate the measurement results according to manufacturer’s instructions by first deducting the overall fluorescence of the animals (unmixing) then assigning regions of interest for autofluorescence (left hind leg with saline) and target fluorescence of inflamed area (right hind leg with zymosan-A).

Wyniki

The encapsulation of high concentrations of fluorescent dyes such as the NIRF dye DY676-COOH used here in the aqueous interior of liposomes leads to a high level of fluorescence quenching. Fluorescence quenching, a phenomenon seen with many fluorophores at high concentration, can be exploited in several in vivo imaging applications where a high sensitivity and reliable detection of the target area are demanded. The use of liposomes also provides protection of the dye which is indispensable for in vivo a...

Dyskusje

Since liposomes can also serve as delivery systems for fluorescent dyes, they enable imaging of target diseases. The encapsulation of high concentrations of fluorescent dyes such as the NIRF dye, DY676-COOH used here, leads to a high level of fluorescence quenching of the entrapped dye. Fluorescence quenching, a phenomenon seen with many fluorophores at high concentration can be exploited in several in vivo imaging applications, where a high sensitivity and reliable detection of the target area is demanded. The ...

Materiały

Name	Company	Catalog Number	Comments
Materials and equipments for preparation of liposomes
egg phospahtidylcholine	Avanti Polar Lipids	840051P	Dissolve in chloroform and store in glass vials (214 mg/ml)
cholesterol	Sigma	C8667	Dissolve in chloroform and store in glass vials (134 mg/ml)
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)	Avanti Polar Lipids	880120P	Dissolve in chloroform and store in glass vials (122 mg/ml)
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)	Avanti Polar Lipids	810145P	Dissolve in chloroform and store in glass vials (2 mg/ml)
Sartorius MC1 (d = 0.01 mg)	Sartorius AG	Research RC 210 P	used for weighing the phospholipids
Rotavapor	Büchi Labortechnik AG	R-114	used for hydration of phospholipid film
Waterbath	Büchi Labortechnik AG	R-481	used for hydration of phospholipid film
Vacuum Controller	Büchi Labortechnik AG	B-720	used for hydration of phospholipid film
Vacobox	Büchi Labortechnik AG	B-177	used for hydration of phospholipid film
Circulation Chiller	LAUDA DR. R. WOBSER GMBH & CO. KG	WKL 230	used for hydration of phospholipid film
DY-676-COOH	Dyomics GmbH	676-00	Dissolve in 10 mM Tris and store stock at -20°C
Tris-(Hydroxymethyl)-aminomethan	Applichem	A1086	buffer 10 mM, pH 7.4
Trichlormethan	Carl Roth GmbH + Co. KG	Y015.2	used for liposome preparation
Sonicator	Merck Eurolab GmbH	USR 170 H	used for liposome preparation
Vortex Genie 2 (Pop-off Cup, No. 146-3011-00)	Scientific Industries Inc.	SI-0256	used for liposome preparation
Sephadex G25 medium	GE Healthcare Europe GmbH	17-0033-01	used for liposome purification
Triton X100	Ferak Berlin GmbH	505002	used to destruct liposomes  for dye quantification
LiposoFast-Basic	Avestin Inc.		used for the extrusion of liposomes
Polycarbonate filter membrane, 100 nm (Whatman Nucleopore Trans Etch Membrane, NUCLEPR PC 19 MM, 0.1 U)	VWR		used for the extrusion of liposomes via LiposoFast-Basic
Fluostar Optima	BMG Labtech		used for dye quantification
Zetasizer Nano ZS	Malvern		used for the determination of liposome size and zetapotential
Ultracentrifuge	Beckmann Coulter GmbH	XL 80	used for concentration of the samples
Rotor	Beckmann Coulter GmbH	SW 55 TI	used for concentration of the samples
Materials and equipments for the evaluation of liposome and optical imaging
Zymosan-A from Saccharomyces cereviciae	Sigma	Z4250-250MG	used for induction of inflammation
Isotonic Saline (0.9%)	Fresenius GmbH	PZN-2159621	used for the dilution of Zymosan-A
Isoflurane vaporizer	Ohmeda Isotec 4		used for anesthesizing animals
Isoflurane	Actavis GmbH	PZN-7253744	anesthesia
Thermo Mat Pro 20 W	Lucky Reptile	61202-HTP-20	used to keep animals warm during anesthesia
Omnican-F (1 ml injection)	Braun	PZN-3115465	used for subcutaneous and intravenous application of probes
Panthenol eye cream	Jenapharm	PZN-3524531	used to prevent dryness of the eyes of animals during anesthesia
Hanks buffered saline solution	PAA Laboratories /Biochrom AG	L2045	w/o Mg2+, Ca2+ and phenol red. For dilution of probes and for washing of cells
8-Well chamber slides	BD Biosciences	354108	used for cell culture followed by microscopy
Cell culture flasks	Greiner BioOne
Cell culture media	Gibco (life technologies GmbH)
Fetal calf serum	Invitrogen
Poly-L-Lysine solution (0.01%, 50 ml)	Sigma	P4832	used to coat cell culture chamber slides
Mountant Permafluor	ThermoScientific	S21022-3	Mounting solution for microscopy
Hoechst-33258	AppliChem		DNA stain for microscopy
Hera-Safe	Heraeus Instruments		sterile work bench used for cell culture
HERA cell	Heraeus Instruments		Incubator used for cell culture
LSM510-Meta	Zeiss		used for confocal microscopy
Maestro-TM in vivo fluorescence imaging system	CRi, Woburn		used for whole body fluorescence imaging of small animals
Spectrophotometer (Ultrospec 4300 pro UV)	GE Healthcare		used for measurement of absorption
Spectrofluorometer (Jasco FP-6200)	Jasco		used for measurement of fluorescence emission
Animals
NMRI mice (8-12 weeks old, male)	Elevage Janvier, France		used for inflammation trials