Cascade 512b

B16 cells cultured on 35-mm dishes were incubated in culture medium containing 3.5 μmol/L FURA-2-AM (Invitrogen) for 1 h at 37 °C, and then rinsed with Hanks’ Balanced Salt Solution (HBSS, Sigma). Each dish was placed into a culture chamber at 37 °C on the stage of an inverted fluorescence microscope (TE2000E, Nikon Instruments, Fi, Italy), connected to a cooled CCD camera (512B Cascade, Roper Scientific, Ottobrunn, Germany). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International, New Jersey, USA) and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image per s) using Metafluor software (Universal Imaging Corporation, Downington PA, USA). Calibration was obtained at the end of each experiment by maximally increasing intracellular Ca²⁺-dependent FURA-2AM fluorescence with 5 μmol/L ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal fluorescence in a Ca²⁺-free medium. [Ca²⁺]_i was calculated according to the formulas previously described54 (link).

For the immunofluorescence assay, the lung sections were treated with rabbit anti-RELM-β polyclonal antibodies (1:200, Abcam, UK) and mouse anti-α-SMA antibody (1:500) or the corresponding vehicle overnight at 4 °C. Then, the sections were incubated with FITC-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:50, Boster, CHN) (excitation: 495 nm, emission: 525 nm) and Cy3-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:50, Boster, CHN) (excitation: 554 nm, emission: 568 nm) for 45 min at room temperature in the dark. Finally, the sections were washed in PBS and mounted with Vectashield hardset mounting medium with 4′,6-diamidino-2-phenylindole dilactate (DAPI, Vector Laboratories, USA). Specimens were observed under a fluorescent inverted microscope (IX73-A22FL/PH; Olympus Corporation; light source: UHP) attached to a CCD digital camera (512B Cascade, Roper Scientific, Tucson, AZ). Blue (WU) and green filters (WIBA) were used to detect FITC (green) and Cy3 (red) signals, respectively. The objectives used were 20 × and 40 × . Images were captured by using the Image-Pro Plus software (version 6.0) and colorized with Adobe Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA).

All the chemicals were of analytical grade and used as received. All solutions were prepared with ultrapure water (18 MΩcm) from a Millipore system. ¹H NMR and ¹³C NMR were acquired in CDCl₃ on BRUKER AVANCE 500 spectrometer using TMS as an internal standard. HRMS were obtained on HP5989 mass spectrometer. The dark-field spectrum measurements were carried out on an inverted microscope (eclipse Ti-U, Nikon, Japan) equipped with a dark field condenser (0.8 < NA < 0.95), a 100 W halogen lamp, a true-color digital camera (Nikon DS-fi), a monochromator (Acton SP2300i) equipped with a spectrograph CCD (CASCADE 512B, Roper Scientific) and a grating (grating density: 300 L/mm; blazed wavelength: 500 nm). The true-color scattering images of gold nanoparticles were taken using a 40X objective lens (NA = 0.8). The scattering spectra from the individual nanoparticles were corrected by subtracting the background spectra taken from the adjacent regions without the GNPs and dividing it with the calibrated response curve of the entire optical system. The spectra were integrated for 10 seconds.

The gold pads were cleaned by immersing them stepwise for 20 s into 100% fuming nitric acid (Merck) and 1 min into a neutralization solution [hydrogen peroxide (30 wt % in water; Merck), ammonia solution (25 wt % in water; Merck) and ddH₂O in the ratio 1:1:5] and rinsed with ddH₂O. Then, such a glass slide was placed in an inverted optical microscope (Axiovert 200M, Carl Zeiss MicroImaging) equipped with a 100×/1.45 numerical aperture oil immersion objective and appropriate fluorescence filter sets. A PDMS ring was sealed with silicon oil on the glass substrate in between the electrodes and contact pads. In addition, the microscope was equipped with micromanipulators (Suess MICROTec PH100) that were used to place tungsten needles (SIGNATONE SE-T) on the contact pads, and thus, connect the electrodes with the function generator (Textonix AFG 320; Sony). The DNA nanostructures were stained with YOYO^®-1 (Life Technologies) in a ratio of 1:10 and diluted with ddH₂O to a final concentration of 45 pM 6HBs and 1.5 mM Mg²⁺. 15 µL of this solution was pipetted into the PDMS ring and an (ac) field applied. Images were taken in the green channel with a frame-transfer intensified CCD camera (Cascade 512:B, Roper Scientific) using the MetaMorph software (Molecular Devices).