2.1 Chemicals and reagents
Resovist® (Ferucarbotran; Schering AG, Berlin, Germany) was the commercial prepared carboxydextran-coated iron oxide nanoparticles that exhibited a monodisperse population of particles with an average diameter to be 58.7 nm, using a particle size analyzer(Shen et al., 2011a). The crystalline core of Resovist® is composed of Fe3O4
and γ-Fe2O3. Resovist® is a solution containing 28 mg Fe/mL and stored at 4°C. For cell culture experiments, Resovist® was diluted to 1, 10, 50, 100 μg Fe/mL with cultured medium.
Anaspec (San Jose, CA): TNF-α converting enzyme (TACE) activity assay kit
Amresco (Solon, OH, USA): ethylene diamine tetraacetic acid (EDTA), sodium chloride (NaCl), sodium phosphate (Na2HPO4)
BD Biosciences (San Jose, California USA): all the antibodies for ELISA assay.
BioLegend (San Diego, CA, USA): anti-mouse CD11b-PE/Cy5, anti-mouse ED-1-APC BioShop (Ontario, Canada): sodium dodecyl sulfate (SDS)
Bio West (Miami, FL, USA): potassium dihydrogen phosphate (KH2PO4), trypan blue Chemicon (Temecula, CA): Hoechst 33258, IL-1β converting enzyme (ICE) protease assay kit
GeneTex (San Antonio, TX): anti-mouse Iba-1 Hayashi (Osaka, Japan): potassium chloride (KCl)
Hyclone (Logan, UT, USA): horse serum, penicillin-streptomycin
Immunochemistry Technologies (Bloomington, MN): Magic Red™ Cathepsin B Assay Kit
Invitrogen (Carlsbad, CA, USA): DQ™ Ovalbumin, E. coli BioParticles-pHrodo, fetal
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bovine serum (FBS), LysoSensor™ Green DND-189, LysoTracker™ Red DND-99, ProLong Gold antifade reagent
Merck (Darmstadt, Germany): acetone, ethanol absolute, paraformaldehyde, sodium azide, sulfuric acid (H2SO4)
Millipore (Billerica, MA,): poly-D-lysine
MP biomedicals (Solon, OH, USA): dimethyl sulfoxide (DMSO)
Serotech (Oxford, UK): FITC- conjugated anti-rabbit IgG secondary antibody
Sigma (St. Louis, MO, USA): 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), acridine orange (AO), albumin bovine serum (BSA), LPS (Escherichia coli O55:B5), sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), Triton X-100, Tween 20
Tocris bioscience (Ellisville, MO, USA): Ac-YVAD-AFC
2.2 Culture of primary murine microglial cells
Primary microglia were prepared from cerebral cortices of 1-3 days old BALB/c mice obtained from the Animal Center of National Taiwan University Hospital. After removing meninges aseptically, neocortical tissues were plated onto 75 cm2 cell culture flasks coated with 25 μg/mL poly-D-lysine. Mixed glial cultures were maintained in DMEM containing 10% heat inactivated FBS, 10% horse serum, 4 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37°C and 5%
CO2 for 12-14 days. Microglia were obtained from the mixed glia cultures by shaking at 165 rpm for 3 h at 37°C. The cell concentration of floating cells was counted using a hemacytometer with trypan blue staining. The floating cells concentration was adjusted to 4×105 cells/mL. According to experimental design, microglia were reseeded onto 24-well (0.5 mL), 48-well (0.3 mL), 96-well culture plate (0.1 mL), 3.5-cm dish (2 mL) and 6-cm dish (5.5 mL) followed by cultured at 37˚C and 5% CO2. After 2 h,
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unattached cells were removed by replacing the medium with DMEM containing 1%
horse serum, 4 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin.
Adherent cells were incubated for 1 h before being used for experiments. More than 85% of the adherent cells were positive for microglia-specific marker CD11b.
2.3 Internalization of iron oxide nanoparticles and colocalized with lysosome Microglia (4 x 105 cells/mL) were cultured on 10-mm coverslips coated with 25 μg/mL poly-D-lysine (0.25 mL/well) and treated with iron oxide nanoparticles (1-100 μg Fe/mL) for 30 min followed by stimulation with LPS (Escherichia coli O55:B5, 100 ng/mL) for 24 h. At the end of incubation, microglia were stained with 1 μM LysoTracker™ Red DND-99 for 2 h at 37°C. After washing twice with PBS (phosphate buffered saline, pH 7.4), cells were fixed with 4% paraformaldehyde for 15 min at room temperature followed by washing with PBS twice. For detection of nuclei, the fixed cells were stained with 5 μg/mL of Hoechst 33258 at room temperature for 5 min. After washing, the cells were mounted in ProLong Gold antifade reagent. Fluorescence confocal images were taken using laser-confocal scanning microscope (Leica TCS SP5 II, Wetzlar, Germany) at emission of 450 nm (Hoechst) and 519 nm (LysoTracker).
2.4 Measurement of cell viability by MTT assay
The viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Microglia
(4 x 105 cells/mL) were cultured in triplicate in 96-well plates (0.1 mL/well) and treated with iron oxide nanoparticles (1-100 μg Fe/mL) for 30 min followed by stimulation with LPS (100 ng/mL) for 24 h. An MTT stock solution (5 mg/mL) was added to the culture (10 μL/well) and incubated for 4 h. The formed formazan was dissolved with a lysis buffer (10% SDS in N,N-dimethylformamide). The absorbance of the formazan product was measured at 570 nm and at 630 nm as background using a microplate
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reader (Dynatech Laboratories, Chantilly, VA, USA).
2.5 Detection of microglial activation marker expression
Microglial cells (4 x 105 cells/mL) were pretreated with iron oxide nanoparticles (1-100 μg Fe/mL) in triplicate for 30 min followed by stimulated with 100 ng/mL LPS for 24 h. The expression of the activation marker of microglia, ionized calcium-binding adaptor molecule-1 (Iba-1) was measured using confocal microscopy and flow cytometry. For confocal imaging, cells were washed with PBS and then fixed in 80%
acetone for 15 min at -20°C. After washing twice, cells were incubated in blocking buffer containing 2.5 % horse serum in PBS for 30 min at 4°C. Cells were stained with rabbit anti-Iba-1 (1:20) in blocking buffer for 1 h at 4°C. After washing twice, the Iba-1 stained cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibody (1:1000) for 30 min at 4°C in the dark. For detection of the nuclei, the cells were stained with Hoechst 33258 (5 μg/mL). After washing, the cells were mounted in ProLong Gold antifade reagent. Fluorescence confocal images were taken using laser-confocal scanning microscope (Leica TCS SP5 II, Wetzlar, Germany) at emission of 450 nm (Hoechst) and 525 nm (FITC).
For flow cytometry, microglia (4 x 105 cells/mL) were cultured in triplicate in a 24-well plate (0.5 mL/well) and treated with iron oxide nanoparticles followed by stimulation with LPS for 24 h as described above. The cells were fixed with 70%
ethanol overnight and stained for the activation marker as described above. After washing, the single cell fluorescence of each sample was measured using a flow cytometry at emission of 525 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
2.6 Analysis of phagocytic activity
The phagocytic activity of microglia was evaluated by using E. coli BioParticles
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conjugated with pHrodo dye following the manufacturer's instructions. Microglial cells were treated with iron oxide nanoparticles as described above in section 2.5. Two hours before the end of incubation, pHrodo E. coli BioParticles (25 μg/μL) were added into each sample. For confocal imaging, cells were fixed and stained with Hoechst 33258 as described above. Fluorescence confocal images were taken using laser-confocal scanning microscope (Leica TCS SP5 II, Wetzlar, Germany) at emission of 450 nm (Hoechst) and 578 nm (pHrodo dye). For flow cytometry, cells were washed twice and single cell fluorescence of each sample was measured using a flow cytometry at emission of 578 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
2.7 Measurement of cytokines by ELISA
Microglial cells (4 x 105 cells/mL) were cultured in triplicate in a 48-well plate (0.3 mL/well) and treated with iron oxide nanoparticles followed by stimulation with LPS for 24 h as described above in section 2.5. The supernatants were collected and quantified for TNF-α and IL-1β by standard sandwich enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated overnight at 4°C with 50 μL per well of anti-mouse TNF-α or IL-1β monoclonal antibody (1:500 dilution in coating buffer; pH 9.5). The wells were washed three times with Tween 20 (0.1%) in PBS and blocked with 200 μL blocking buffer (1 % bovine serum albumin (BSA) in PBS) at room temperature for 1 h. After washing, 50 μL of samples and standards diluted with blocking buffer were added and incubated at room temperature for 2 hrs. After washing, biotinylated anti-mouse TNF-α or IL-1β monoclonal antibody (TNF-α: 1:500;
IL-1β: 1:2000 in blocking buffer) was added (50 μL/well) and incubated at room temperature for 1 h. After washing, streptavidin-HRP conjugate (TNF-α: 1:250; IL-1β:
1:125 in blocking buffer) was added (50 μL/well) and incubated at room temperature
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for 30 min. After washing, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added (50 μL/well) to detect bound peroxidase conjugate. Finally, 150 μL stop solution (3N H2SO4) were added into each well to terminate the reaction. The optical density (O.D.) was measured at 450 nm using a microplate reader (Dynatech Laboratories, Chantilly, VA, USA).
2.8 Measurement of IL-1β converting enzyme activity using enzymatic assay
The IL-1β converting enzyme (ICE) activity was measured spectrophotometrically with a commercial ICE protease assay kit using ICE fluorogenic substrates Ac-YVAD-AFC. Microglial cells (4 x 105 cells/mL) were cultured in a 6-cm dish (5.5 mL/dish) and treated with iron oxide nanoparticles followed by stimulation with LPS for 24 h as described above. After washing twice, the cells were scraped and dissolved with 25 μL lysis buffer for 10 min at 4°C. After centrifugation (10,000 x g, 1 min, 4°C) the supernatants were collected. For measurement ICE activity, 25μL of sample dissolved with lysis buffer was incubated with 25 μL of diluted ICE substrates (200 μM in lysis buffer) and DTT (5 mM in lysis buffer) in a 96-well black plate for 1 h at 37°C in the dark. The absorbance in the wells was detected at 505 nm after excited by 400 nm, using a fluorescence microplate reader (Dynatech Laboratories, Chantilly, VA, USA).
2.9 Measurement of TNF-α converting enzyme activity using enzymatic assay
The TNF-α converting enzyme (TACE) activity was measured spectrophotometrically with a commercial TACE activity assay kit. Microglial cells (4 x 105 cells/mL) were cultured in a 3.5-cm dish (2 mL/dish) and treated with iron oxide nanoparticles followed by stimulation with LPS for 24 h as described above. After washing twice, the cells were scraped and dissolved with 50 μL lysis buffer (0.1%
Triton X-100 in assay buffer). After centrifugation (2,500 x g, 10 min, 4°C) the supernatants were collected. For measurement TACE activity, 25 μL of sample or
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standards diluted with lysis buffer was incubated with 25 μL of diluted TACE substrates (40 μM in assay buffer) in a 96-well black plate for 1 h at 37°C in the dark, and then 25 μL of stop solutions were added into each well to terminate the reaction. The absorbance in the wells was detected at 520 nm after excited by 490 nm, using a fluorescence microplate reader (Dynatech Laboratories, Chantilly, VA, USA).
2.10 Analysis of amount of lysosomes
The total lysosomal number of microglia was evaluated by ectodermal dysplasia-1 (ED-1) expression that was determined using confocal microscopy and flow cytometry.
Microglial cells were treated with iron oxide nanoparticles, fixed and blocked as described above in section 2.5. The cells were stained with allophycocyanin (APC)-labeled rat anti-ED-1 antibody (1 μg/106 cells ) in blocking buffer for 1 h at 4°C in the dark. For confocal imaging, cells were also stained with Hoechst 33258 for imaging nuclei. Fluorescence confocal images were taken using a laser-confocal scanning microscope (Leica TCS SP5 II, Wetzlar, Germany) at emission of 450 nm (Hoechst 33258) and 670 nm (APC). For flow cytometry, the single cell fluorescence of each sample was measured using a flow cytometry at emission of 670 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
2.11 Measurement of lysosomal membrane permeability
The lysosomal permeability of microglia was measured with acridine orange (AO) staining by flow cytometry. In brief, microglial cells were treated with iron oxide nanoparticles as described above. At the end of incubation, the cells were stained with 5 μg/mL AO for 10 min at 37°C. After washing, the single cell fluorescence of each sample was measured using a flow cytometry at emission of 617 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
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2.12 Measurement of lysosomal pH
The lysosomal pH of microglia was measured with LysoSensor™ Green DND-189 staining by confocal microscopy and flow cytometry. Microglial cells were treated with iron oxide nanoparticles as described above. At the end of incubation, the cells were stained with 1 μM LysoSensor for 2 h at 37°C. For confocal imaging, cells were also stained with Hoechst 33258. Fluorescence confocal images were taken using a laser-confocal scanning microscope (Leica TCS SP5 II, Wetzlar, Germany) at emission of 450 nm (Hoechst 33258) and 519 nm (LysoSensor). For flow cytometry, the single cell fluorescence of each sample was measured using a flow cytometry at emission of 519 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
2.13 Measurement of proteolytic activity
The proteolytic activity of microglia was determined using DQ™ Ovalbumin (DQ-OVA) by confocal microscopy and flow cytometry. Microglial cells were treated with iron oxide nanoparticles as described above. At the end of incubation, the cells were stained with 50 μg/mL DQ-OVA for 1 h at 37°C. For confocal imaging, cells were fixed and stained with Hoechst 33258. Fluorescence confocal images were taken using a laser-confocal scanning microscope (Leica TCS SP5 II, Wetzlar, Germany) at emission of 450 nm (Hoechst 33258) and 510 nm (DQ-OVA). For flow cytometry, the single cell fluorescence of each sample was measured using a flow cytometry at emission of 519 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
2.14 Measurement of cathepsin B activity
The cathepsin B activity of microglia was measured using Magic Red™ cathepsin B assay kit by flow cytometry following the supplier’s instructions. In brief, microglial
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cells were treated with iron oxide nanoparticles as described above. At the end of incubation, the cells were stained with cell-permeable cathepsin B substrate for 1 h at 37°C. After washing, the single cell fluorescence of each sample was measured using a flow cytometry at emission of 617 nm (BD LSRFortessa, San Jose, CA). The data were analyzed using the software Flowjo 5.7.
2.15 Statistical analysis
The data were expressed as mean ± standard error (SE) for each treatment group in the individual experiments. Statistical differences between groups were evaluated by one-way ANOVA and Student’s t-test was used to compare treatment groups to the vehicle control when significant differences were observed. *, p < 0.05 was defined as statistically significant.
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