functional role in cell migration, lymphocyte homing and adhesion during hematopoiesis and lymphocyte activation [5,6]. CD45 or leukocyte common antigen is present on all human leukocytes [7] and on the surface of 85% to 95% of both B-cell lymphoma and leukemic cells [8]. So we tested mouse anti-human mAbs against these two markers. Anti-CD45 mAb is already used in clinic for immunoradiotherapy to target a radioisotope to tumor cells [9–11]. Therefore, in order to establish a diagnostic tool to detect leukemic foci and to perform staging of the disease in mouse models, we generated two fluorescent antibodies. We first validated this methodology by using an in vivo model of luminescent human leukemia HL60-Luc which expresses both hCD44 and hCD45 to compare bioluminescent imaging (BLI, tumor cells) and fluorescence reflectance imaging (FRI, mAb). We then applied this method on leukemic cells from patient samples in vivo. Methods Reagents Antibody labeling kit containing AF750 dye (SAIVITM Rapid Antibody Labeling kits) was purchased from Invitrogen (Cergy- Pontoise, France). The following mAbs were used: purified NA/ LE mouse IgG1 anti-human CD44, clone 515 and NA/LE mouse IgG1 anti-human CD45, clone HI30 (BD Biosciences, Le Pont de Claix, France). This anti-human CD44 mAb recognizes all the isoforms of the hCD44 cell surface glycoprotein and the HI30 CD45 mAb reacts with all isoforms of human CD45. Labeling of antibodies The antibodies were labeled according to the manufacturer’s instructions. The protein concentration and the degree of labeling were calculated with the absorptions at 280nm (protein) and 752nm (dye). Cells The human leukemia cell line HL60 purchased from the ATCC (Manassas, VA, USA) was stably transfected with the firefly luciferase reporter gene and clonally selected to generate the HL60-Luc cell line [12]. This cell line was grown in RPMI 1640 supplemented with 20% foetal calf serum, 100 units/ml streptomycin, 100 units/ml penicillin and 1.25 mg/ml fungizone. Immediately before implantation in NOD/SCID mice, cells were centrifugated and adjusted to a concentration of 256106 cells per ml in PBS for intravenous injection. Cells were maintained in a 5% CO2 humidified atmosphere at 37uC. Materials for cell culture were obtained from Gibco (Cergy-Pontoise, France). The human colorectal carcinoma cell line HCT116-Luc was purchased from Caliper (USA). This cell line was grown in MEM supplemented with 20% foetal calf serum, glutamine 100 units/ml streptomycin and 100 units/ml penicillin. Cells were tested negative for mycoplasma or bacterial contamination by an independent laboratory (Clean-cells S.A., Bouffe´re´, France). The studies were approved by the scientific committee of the Centre de Ressources Biologiques des He´mopathies Malignes (INSERM Midi-Pyre´ne´es). For human samples, written consent was obtained and all the data were analysed anonymously. Diagnosis was made using cytomorphology, cytogenetics and leucocyte antigen expression and evaluated according to the French-American-British (FAB) classification. The characteristics of this sample were: FAB5, normal karyotype, FLT-ITD NPM mutated. The phenotypic characteristics of this sample before grafting into NOD/SCID mice were: 92.1% of CD45+, 89.8% of CD33+, 5.1% of CD34+, 11.3% of CD38+ and 10.1% of Optical Imaging of Leukemia CD123+, and after grafting were: 85.3% of CD45+, 89.6% of CD33+, 1.7% of CD34+, 62.9% of CD38+ and 1.8% of CD123+. Cell binding Cell binding studies with the AF750-mAb at a single concentration of mAb (5 mg/ml) were carried out in triplicate using HL60-Luc cells (16106 cells per experiment) and HCT116- Luc cells (negative control, does not express CD45, 16106 cells per experiment). Cells were incubated in the presence of the AF750- mAb for 2h at room temperature. For control or nonspecific uptake experiments the cells were first saturated by incubating with an excess of non-fluorescent mAb (100 mg/ml) for 0.5 h. The total fluorescence was measured with an IVIS Lumina II (Caliper, USA) after which samples were centrifuged and washed twice in PBS. Student’s t test (p,0.05) was used to determine statistical differences in the cell binding of the fluorescent mAbs. Animal tumor model Homozygous female NonObese Diabetic/Severe Combined ImmunoDefiency (NOD/SCID) mice (NOD.CB17-Prkdcscid/J, Charles River Laboratories, Saint-Germain-sur-L’Arbresle, France), were used for implantation of human AML cells. These mice have a severe combined immunodeficiency affecting T- and B-lymphocyte development and a natural killer cell functional deficit, so they are adapted to the engraftment of human cells. Animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the European Directive EEC/86/609, under the supervision of authorized investigatorsAll mice received sublethal irradiation 24 hours before either i.v. transplantation with either 56106 HL60-Luc cells or 26106 patient AML cells. The experiments were performed 7 to 30 days after leukemia cell injection. Bioluminescence imaging Animals were peritoneally injected with 125mg/kg luciferin potassium salt (Caliper Life Sciences, Roissy, France) before undergoing anesthesia with 3% isoflurane (AerraneH, Maurepas, France) in air in an anesthesia induction box. Then 2% isoflurane in air/O2 was continuously delivered via a nose cone system in the dark box of a high sensitivity CCD camera cooled to 290uC (IVIS Lumina II, Caliper, USA). BLI was performed 10 minutes after substrate injection. Acquisition setting (binning and duration) were set up depending upon tumor activity at the time of acquisition. Fluorescence imaging Fluorescent mAb was injected via the tail vein into HL60-Luc tumor-bearing mice. After 24h, mice were anesthetized then fluorescent images were obtained (after fur removal) with the Lumina II (Caliper) using one filter set (excitation: 745nm, emission: 810nm). Acquisition setting (binning and duration) were set up depending upon tumor activity at the time of acquisition. Determination of the minimal fluorescent monoclonal antibodies dose required and effects of fluorescent monoclonal antibodies on in vivo leukemia proliferation Several doses of mAbs, between 1 and 10 mg were intravenously injected to leukemia-bearing mice. Mice were imaged 24 and 48h after using first BLI to locate the tumor foci, and then using FRI. The images were then compared to determine if all biolumines- cent foci were revealed with the fluorescent mAbs and the colocalization was assessed by the calculation of Pearson correlation coefficient (ImageJ software). PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30690