Wheat germ agglutinin alexa fluor 633 conjugate

The care of the animals and all procedures used in these experiments were approved by the Committee on Animal Experiments of Graduate School of Bioagricultural Sciences, Nagoya University. Spleens were isolated from C57BL/6 mice (6 weeks, female, Japan SLC, Inc., Hamamatsu, Japan), and splenocytes were prepared (Additional file 1). Splenic DCs (approximately 2.0 × 10⁵ cells) were mixed with CF488A-labeled α-DC-ZZ-BNC-OVA (2 μg as OVA), incubated in RPMI1640 medium supplemented with 10% (v/v) FBS at 37 °C for 4 h. The cells were washed and analyzed by a flow cytometer BD FACS Canto II (BD Biosciences, San Jose, CA, USA). Splenic DCs loaded with CF488A-labeled α-DC-ZZ-BNC-OVA were stained with Wheat Germ Agglutinin Alexa Fluor 633 Conjugate (Life Technologies). The cells were observed under a confocal laser scanning microscopy (LSM) model FV-1000D (Olympus, Tokyo, Japan). Whole cell Z-stacks (each slice = 0.5 μm, total 14 sections) were acquired by LSM, which was equipped with a × 100 oil objective lens (Olympus).

H295R cells were cultured on sterilized and poly l-lysine coated cover slips for 24 hours. After lipofectamine transfection, cell plasma membranes were stained with Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate (W21404, Life Technologies) for 10 minutes at 37°C. Cells were washed twice with PBS (5 minute each), fixed with 4% paraformaldehyde and permeabilized with 1% trition-X100 in PBS, 10 minutes each at room temperature. Finally, cells were washed 3× in PBS, and cover slips were mounted on slides using Vectashield Antifade Mounting Medium with DAPI (H-1200, Vector Laboratories). Confocal images were taken using Zeiss LSM510 Meta confocal microscope and analyzed using Zen 2011 software.

Experiments to investigate the intracellular localization of NPs were performed in Lab 2 (Mainz) and Lab 3 (Jena) using confocal laser scanning microscopy (CLSM). For these experiments the HepG2 cells were seeded in ibiTreat µ-slides (IBIDI, Germany) (Lab 2, Mainz) or in glass chamber slides (BD, Germany) (Lab 3, Jena). After 24 h, NPs were added to the medium at a concentration up to 500 µg/mL. Before CLSM imaging cells were washed with PBS. Live cell images were taken in Lab 2 (Mainz) with a commercial setup (LSM SP5 STED Leica Laser Scanning Confocal Microscope, Leica). There, the cell membranes were stained with CellMaskOrange (2.5 mg·mL⁻¹, Invitrogen), and the cell nucleus with DraQ5 (2.5·10⁻⁶ M, Biostatus). In Lab 3 (Jena), for microscopy observations with the LSM 510 Meta (Carl Zeiss MicroImaging GmbH) cell membrane was stained with Wheat Germ Agglutinin Alexa Fluor® 633 conjugate (Invitrogen) and the nucleus with Hoechst 33258 (Applichem) after fixation of cells with 4% formaldehyde. Fluorescent particles were detected at 533–570 nm. For a control experiment to investigate the clustering and adhesion of the NPs to differently coated culture slides, the SiO₂ NPs were suspended in complete culture medium by pipetting and then they were added to the slides without any cells for supplementary microscopy studies.