Clodronate liposome

All animal experiments were conducted using institutionally approved protocols (Institutional Animal Care and Use Committee). C57BL/6 mice (female unless otherwise indicated) were obtained from Charles River Laboratories, Wilmington, MA. STZ mice (males) and NOD and NOD/SCID mice (females) were obtained from the Jackson Laboratory (Bar Harbor, ME). STZ mice exhibited hyperglycemia (blood glucose > 250 mg/dl) upon arrival. In experiments involving clodronate liposomes, clodronate liposomes and control liposomes were purchased from Liposoma (Amsterdam, The Netherlands). Mice received intraperitoneal injections of 200 μl of clodronate or control liposomes 48 hours before LNP treatment. For fluorescence and luminescence studies, dissected organs were imaged using an IVIS (Perkin Elmer). In experiments using mRNA encoding luciferase, mice received an intraperitoneal injection of 130 μl of d-luciferin (30 mg/ml) 15 min before imaging. Blood samples were drawn via submandibular bleed and collected in Microtainer Serum Separator tubes (Becton Dickinson, Franklin Lakes, NJ).

Clodronate liposomes (Liposoma BV, Amsterdam, The Netherlands) or PBS control liposomes were injected into NMRI mice (n = 6 mice; Clodronate liposomes n = 6 joints; PBS control liposomes n = 6 joints) both on the knee (i.a. 20 µL/joint) and tail (i.v. 200 µL/mouse) for eliminating monocytes/macrophages [63 (link)] one day before testing with Newman EVs (20 µg in 20 µL).

For the monocyte infiltration into atherosclerotic lesion assay, experimental male mice were fed a WD for 10 weeks. Clodronate-liposomes (250 μl, Liposoma) were i.v. injected in order to transiently deplete monocytes, followed by i.v. injection of 250 μl fluorescent microspheres 48 h later. Fluoresbrite FITC-dyed (YG, 0.5 μm) plain microspheres (2.5% solids [w/v]; Polysciences) were diluted 1:25 in PBS^{26 (link),27 (link)}. Mice were euthanized and hearts with aortic root was then used for consecutive sections from the atrioventricular valve at a thickness of 20 μm. Nuclei were counter-stained by DAPI Fluor mount-G (SouthernBiotech). Images were then captured using a fluorescence microscope (Carl Zeiss). Beads that reflect monocyte recruitment were quantified in 3–5 aortic sinus sections per mouse.
For the murine peritoneal mono-macrophage recruitment, 1 ml of sterile 4% thioglycolate media was injected intra-peritoneally. The cells from murine peritoneal cavities were harvested 3 days later, and analyzed by cell counter or flow cytometry.

Neutrophils were depleted from mice by a single intraperitoneal injection of 500 μg/body anti-Ly6G mAb (1A8 clone; Bio X cell, USA)^{28 (link)}. Rat IgG2a Isotype control (2A3 clone; Bio X cell) was injected into control mice. Macrophages were depleted by a single intravenous injection of 10 μL/g clodronate liposomes (Liposoma B.V., Netherlands)^{29 (link)}. Control liposomes were intravenously administered to control mice. The depletion of neutrophils or macrophages was confirmed by flow cytometry. In flow cytometry, neutrophils and macrophages were regarded as Gr-1^high CD11b⁺ F4/80⁻ cells and F4/80⁺ CD11b^int Gr-1⁻ cells, respectively. Cells were isolated from whole blood collected from the right atrium of mice, aliquoted at 5 × 10⁵ cells/tube, and pre-treated with mouse serum prior to staining. Cells were then stained with the following antibodies: anti-CD11b-FITC (Rat IgG2b kappa, M1/70 clone; BD Biosciences, USA), anti-F4/80-APC (Rat IgG2b kappa, BM8.1 clone; Tonbo Biosciences, USA), and anti-GR1-PE (Rat IgG2b kappa, RB6–8C5 clone; Tonbo Biosciences). Dead cells were excluded using 7-AAD (BD Biosciences). Stained cells were run on BD FACS Aria (BD Biosciences) and analyzed using FlowJo software (BD Biosciences). In infection experiments, mice were infected with GAS 24 h after administration of each depletion or control agent.

Spleens were smashed, total splenocytes were loaded on MACS columns and the magnetic positive fraction was stained for F4/80 and CD11b followed by FACS isolation [18 ].
For pharmacologic RPM depletion, BL/6 CML mice were treated with 1 ml of clodronate liposomes (equal 5 mg of clodronate) (Liposoma, the Netherlands (clodronateliposomes.com)) or vehicle (PBS) every 5^th day by intraperitoneal injection starting 3 days prior to CML induction.

WT, Alox15^−/− or Ccr2^−/− mice were treated with control or clodronate liposomes (70 µl, intranasally; Liposoma BV). The AM populations were evaluated at day 2 and day 14 after delivery in the BAL by flow cytometry.