Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in stay single cells by site-specific direct stochastic optical reconstruction microscopy | Journal of Nanobiotechnology


Coverslip preparation for dSTORM imaging

No. 1 (thickness, 0.13–0.16 mm) Corning glass coverslips with dimensions 22 × 22 mm (Paul Marienfeld GmbH & Co. KG, Lauda-Konigshofen, Germany) have been washed with deionized water (Human Energy 1 + , Human Company, Seoul, Korea) and spectroscopic-grade ethanol (Duksan Pure Chemical Co. Ltd., Ansan, Korea), adopted once more by washing with ultrapure water and spectroscopic-grade methanol (Duksan) earlier than air-drying below UV mild for 30 min. A combination of 10 µg/mL poly-L-lysine (PLL, Sigma-Aldrich, St. Louis, MO, USA) and 20 µg/mL laminin (Sigma-Aldrich, St. Louis, MO, USA) was coated on the precleaned coverslips and incubated for 3 h at room temperature.

Cell tradition and labeling with MNPs@SiO2(RITC)

RAW 264.7, HEK293, and NIH3T3 cells have been maintained in T25 plastic tissue tradition dishes containing DMEM (Gibco, 12800-058, Grand Island, NY, USA) with 1% antibiotic–antimycotic agent (Gibco, 15240-062, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; Gibco, 26140-079, Grand Island, NY, USA) at 37 °C in a humidified 5% CO2 incubator. Each two days, HEK293 and NIH3T3 cells have been subcultured by trypsinization, and RAW 264.7 cells have been subcultured with a cell lifter. The cells have been plated on PLL/laminin-coated glass coverslips and incubated in a single day in a humidified incubator at 37 °C. MNPs@SiO2(RITC) consisted of a ~ 9-nm cobalt ferrite core (CoFe2O3) and a RITC-incorporating silica shell [3] have been bought from Biterials (Seoul, South Korea). The scale of MNPs@SiO2(RITC) is 50 nm, and zeta potential is between − 40 and − 30 mV [3, 34]. Rhodamine B containing SiO2 NPs (10-nm, 50-nm, and 200-nm measurement) have been bought from CD Bioparticles (Shirley, NY, USA). We confirmed the construction of the MNPs@SiO2(RITC) with X-ray diffraction (XRD) utilizing a Excessive-Energy X-Ray Diffractometer (Ultima III, Rigaku, Japan), which confirmed particular patterns of CoFe2O4 and amorphous silica beads (information not proven). The cells have been washed thrice with phosphate-buffered saline (PBS) and labeled with the specified focus of MNPs@SiO2(RITC) and SiO2 NPs in FBS-free DMEM. Earlier than information acquisition, extra NPs have been squeezed out and the cells have been washed thrice with PBS. The coverslips with NP-labeled cells have been positioned on the dove prism and dSTORM resolution [10 mM β-mercaptoethylamine (MEA), 0.5 mg/mL glucose oxidase, 40 μg/mL catalase, 10% glucose (Sigma-Aldrich, St. Louis, MO, USA)] was added on prime of the cells, which have been incubated for 15 min previous to imaging.

Main liver cells have been remoted from 8-week-old male C57BL/6 mice (Doo Yeol Biotech, Seoul, Korea), as beforehand described [35,36,37]. Experimental protocols have been accepted by the Laboratory Animal Analysis Heart of Ajou College Medical Heart. Briefly, mice have been anesthetized utilizing isoflurane previous to the belly operation. The liver was perfused with perfusion buffer (10 mM HEPES, 140 mM NaCl, 6 mM KCl, and a couple of mM EGTA, pH 7.4) for five min at a movement fee of seven mL/min. The liver was then re-perfused with collagenase buffer [0.48 mM HEPES, 66.7 mM NaCl, 6.7 mM KCl, 0.48 mM CaCl2, and 0.8 mg/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA), pH 7.4] and extracted from the mouse physique. The extracted liver was homogenized, and first hepatocytes have been remoted by centrifugation at 50×g for five min and layered utilizing a 40% Percoll cushion by centrifugation at 300×g for 7 min. The remoted hepatocytes have been incubated on plates and canopy glasses coated with collagen sort I (Sigma-Aldrich, St. Louis, MO, USA) in development medium [Medium 199 (Thermo Fisher Scientific, Sunnyvale, CA, USA) with 10% FBS and 1% antibiotic–antimycotic agent]. Liver non-parenchymal cells fraction, containing Kupffer cells, liver sinusoidal endothelial cells, and stellate cells, was collected from the homogenized liver by layering with 25% and 50% Percoll gradient utilizing centrifugation at 1800×g for 30 min. The layer, containing Kupffer cells and liver sinusoidal endothelial cells, was separated into Kupffer cells and liver sinusoidal endothelial cells by selective adhesion [37]. The layer was transferred to a tradition plate and canopy glass with RPMI 1640 media (Thermo Fisher Scientific, Sunnyvale, CA, USA) containing 10% FBS and 1% antibiotic–antimycotic agent and incubated for 8 min at room temperature to make sure adhesion of Kupffer cells onto the plate and canopy glass. Non-adhered cells (liver sinusoidal endothelial cells) have been collected and transferred to a collagen sort I-coated plate and canopy glass containing DMEM (Gibco, Grand Island, NY, USA) with 10% FBS and 1% antibiotic–antimycotic agent. The three kinds of cells have been incubated at 37 °C in a humidified 5% CO2 incubator. The remoted mouse main hepatocytes, Kupffer cells and liver sinusoidal endothelial cells have been characterised based mostly on morphological evaluation and particular marker protein expression: albumin for hepatocytes, F4/80 [encoded in adhesion G protein coupled receptor E1 (ADGRE1) gene] for Kupffer cells, platelet endothelial cell adhesion molecule [PECAM-1, also known as cluster of differentiation 31 (CD31)] for liver sinusoidal endothelial cells (Extra file 1: Fig. S1).

dSTORM setup and information acquisition

A lab-made dove prism-type TIRF setup, resembling the equipment beforehand utilized in our group, however with some modifications, was employed (Extra file 1: Fig. S2) [38]. The setup consisted of an Olympus BX53 upright microscope (Olympus Optical Co., Ltd., Tokyo, Japan) geared up with a × 100-oil sort goal lens with numerical aperture 1.4 (Olympus, UPlanSApo) and a DIC slider with Nomarski prism (Olympus, U-DICT). The dove prism with the pattern was positioned on the prism holder. Gentle sources from 532-nm-(Changchun New Industries Optoelectronics Tech. CO., Ltd., Jilin, China) and 405-nm-wavelength lasers (COMPACT-30G-405, World Star Tech., Toronto, ON, Canada) have been directed to the prism with the assistance of mirrors to acquire complete inside reflection and to provide an evanescent subject layer on the interface. An electron-multiplying cooled charge-coupled system digicam (EM-CCD, 512 × 512 pixels imaging array, iXon Extremely, Andor, Belfast, Eire), together with a 575/15 nm band-pass filter (Semrock, Rochester, NY, USA) positioned on the optical path of the microscope, was used to amass the fluorescence photographs. MetaMorph software program (Molecular Units, LLC, Sunnyvale, CA, USA) was used to manage the picture acquisition, shutter pace, and publicity time of the digicam.

dSTORM picture evaluation

Acquired photographs have been analyzed utilizing a ThunderStorm plugin in ImageJ (http://rsbweb.nih.gov/ij/) software program. For superlocalization, the centroid of the fluorescence spot was fitted to the two-dimensional (2D) Gaussian perform [39]:

$$fleft(x, y,{z}_{o},A,{x}_{o},{y}_{o},{s}_{x},{s}_{y}proper)={z}_{o}+A expleft[-frac{1}{2}left[{left(frac{(x-{x}_{o}}{{s}_{x}}right)}^{2}-{left(frac{y-{y}_{o}}{{s}_{y}}right)}^{2}right]proper]$$

the place sx and sy are the usual deviations alongside instructions x and y, respectively, xo and yo are coordinates of the facilities, zo is a continuing from background noise, and A is the amplitude. Localization precision σ [40] was calculated with.

$${varvec{sigma}}=sqrt{left(frac{{s}^{2}}{N}+frac{{a}^{2}}{12N}+frac{8pi {s}^{4}{b}^{2}}{{a}^{2}{N}^{2}}proper),}$$

the place N is the variety of collected photons, a is the pixel measurement of the detector, s is the usual deviation of the point-spread perform, and b is the background noise of the detector. From the reconstructed dSTORM photographs, the person NPs have been recognized in massive fields of view, and their properties have been assessed with a becoming process. By the technique of aggregated information set and imply worth of the fitted spots, particular person NPs have been investigated and quantified. The dSTORM photographs of stay cells have been drift-corrected utilizing the brilliant spots on the coverslips as reference markers. The brilliant spots on coverslips have been used as fiducial markers. The picture contained stay cell and fiducial marker was acquired. The acquired photographs have been reconstructed by ThunderSTORM to acquire the NP distribution in cells. After superlocalization, the picture of fiducial marker was loaded and drift correction was carried out utilizing ThunderSTORM robotically (Extra file 1: Fig. S3) [41]. The NP quantity in cells was counted by TrackMate, a plugin of ImageJ (NIH, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/).

Cell viability assay

For the cell viability assay, an MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay was carried out as described in a earlier examine [21, 42, 43], utilizing a CellTiter 96-cell proliferation assay equipment (Promega Company, Madison, WI, USA) in keeping with the producer’s directions. On this step, 2 × 104 cells have been seeded on a 96-well assay plate and handled with MNPs@SiO2(RITC) and SiO2 NPs for 12 h. MTS resolution was added to every effectively of the 96-well assay plate containing handled cells in 100 µL of tradition medium. The assay plate was then incubated for 1 h below 5% CO2 at 37 °C. The quantity of soluble formazan produced by mobile discount was immediately measured with a plate reader at a wavelength of 490 nm. The values have been normalized relative to the protein optical density worth for every corresponding group.

Microarray evaluation

Gene expression profiles have been analyzed as described in a earlier examine [44,45,46,47]. On this step, modifications in gene expressions of MNPs@SiO2(RITC)-treated cells (0.1 mg/mL, 1.0 mg/mL) have been examined utilizing a Human U133 Plus 2.0 50 Okay Affymetrix system (ISTECH Corp., Korea), which accommodates 54,675 probes. Organic pathways have been enriched utilizing the web-based bioinformatics software program Ingenuity Pathway Evaluation (IPA; Qiagen, Valencia, CA, USA). A threefold change in gene expression was used as a cut-off to generate information units of considerably modified genes.

Actual-time polymerase chain response (PCR)

The expressions of DNA repair-related genes have been decided by utilizing a real-time PCR equipment, based mostly on RealMOD™ SYBR Inexperienced (Intron, Seong-Nam, Korea), that employed Rotor Gene-Q (Qiagen, Valencia, CA) with gene-specific primer pairs (Extra file 1: Desk S1). The PCR reactions have been carried out at 95 °C for five min adopted by 50 cycles of 95 °C for five s and 60 °C for 30 s, in keeping with the producer’s protocol. The edge/quantification cycle (Ct/Cq) worth was decided on the level by which fluorescence was detected statistically above the background, and PCR merchandise have been analyzed by producing a melting curve, constructed by Rotor-Gene 1.7 software program (Qiagen, Valencia, CA). PCR was run in unbiased triplicates. The relative quantification of those gene expressions was calculated by the two−ddCt technique.

Quantification of micronuclei

Cells have been cultured in a 24-well plate (SPL, Suwon, Korea) and handled with MNPs@SiO2(RITC) for 12 h. The cells have been then stained with 10 µg/mL of Hoechst33342 (Invitrogen, Carlsbad, CA, USA) in PBS for 10 min at room temperature and washed thrice with PBS. Fluorescent photographs have been acquired by Axio Vert 200 M fluorescence microscopy (Zeiss, Jena, Germany) on the Three-Dimensional Immune System Imaging Core Facility of Ajou College. The excitation wavelengths for Hoechst33342 and MNPs@SiO2(RITC) have been 405 and 530 nm, respectively. Micronuclei have been counted in 5 to seven randomly chosen areas on every effectively. Greater than 200 cells per experimental group have been counted utilizing ImageJ. The statistical evaluation was completed by assessing the outcomes by one-way evaluation of variance (ANOVA) with Bonferroni’s multiple-comparison checks utilizing IBM-SPSS software program (IBM Corp., USA). In all analyses, p < 0.05 was taken to point statistical significance.

Inductively coupled plasma/quadrupole mass spectrometer (ICP-QMS) and atomic absorption spectrometer (AAS) evaluation

An inductively coupled plasma/quadrupole mass spectrometer (PerkinElmer, MA, USA) was used for the quantitative estimation of Co. The evaluation was completed with a 1600 W RF energy provide, 18.0 L/min coolant gas-flow fee, 1.30 L/min auxiliary gas-flow fee, and 0.98 L/min nebulizer gas-flow fee. For quantitative estimation of Fe, an atomic absorption spectrometer (Thermo Fischer, MA, USA) was used together with a flame atomizer system and an air-acetylene gasoline with a 1.0 L/min gasoline movement at a 248.3 nm wavelength.

The nucleus fraction was ready with Nuclei EZ™ prep equipment (Sigma-Aldrich, St. Louis, MO, USA). Isolation procedures have been carried out in keeping with the producer’s directions. On this step, 3 × 106 cells have been washed with ice-cold PBS and lysed with Nuclei EZ lysis buffer. The cells have been harvested with a scraper and incubated for five min on ice. The lysed fractions have been pelleted by centrifugation at 500 g for five min at 4 °C. The pellets have been washed with Nuclei EZ lysis buffer and vortexed. Ultimate nuclei fractions have been collected by centrifugation at 500g for five min at 4 °C and resuspended with a Nuclei EZ storage buffer.


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