Leica tcs sp5 2 microscope

Giant unilamellar vesicles (GUVs) were produced following the electro-formation method as described in previous works (Apellaniz et al., 2010). A total of 2 mM of lipid (POPC or POPC:POPS (1:1)) was dissolved in 200 μL CHCl₃:CH₃OH with the fluorescent probe NBD-PE (0.5%). When required, MPER-TMD_671–709 peptide (dissolved in 10% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)) was included in the organic phase at 1:250 peptide-to-lipid ratio. The GUVs were transferred to a BSA-blocked microscope chamber and incubated for 15 min with 250 nM of 10E8-STAR RED fluorescent Fabs. The images were acquired on a Leica TCS SP5 II microscope (Leica Microsystems GmbH, Wetzlar, Germany). NBD-stained GUVs were excited at 476 nm, and emission was imaged at 530 ± 20 nm by using a 63× water immersion objective (numerical aperture (NA) = 1.2). The STAR RED-labeled Fab fragments were excited at 633 nm by using a HeNe laser, and emission was imaged at 775 ± 125 nm. Relative extents of Fab–GUV binding were obtained by measuring the fluorescence intensity of STAR RED along the equatorial plane of the GUV images, and normalized with WT values as the reference, in a number of vesicles n ≥ 8 and n ≥ 17 in lipid alone and lipid–peptide samples, respectively.

NF-κB is a transcription factor which regulates many physiological processes including cell death, inflammation, cell proliferation and immune response [16 (link)]. NF-κB protects the cells from undergoing apoptosis as a response to DNA damage or cytokines. Regulation of the signaling pathways of NF-κB activity plays an essential role in cancer development and progression [16 (link)]. NF-κB kit (Thermo Fisher Scientific, Pittsburgh, PA, USA) was used for this assay. Briefly, HepG2 cells were seeded overnight at a density of 4 × 10⁴ in 12 well-plates covered by a cover slide. Then cells were treated with the IC₅₀ of CADMN for 24 h, 48 h and 72 h and then induced by 100 ng/mL TNF-α (Santa Cruz, CA, USA) for 24 h. After that, cells were fixed and stained according to the protocol provided by the manufacturer and analyzed using confocal Leica TCS SP5 II microscope (Leica Microsystems, Mannheim, Germany). The Intensity of cytoplasmic and nuclear NF-κB was measured using Image J software (NIH, Bethesda, MD, USA). The average intensity of 35 objects/sample was quantified. The ratios were then compared among TNF-α-stimulated, CADMN-treated, and untreated cells [14 (link)]. 5-FU was used as a positive control.

Static images and videos were acquired on a Leica TCS SP5 II microscope (Leica Microsystems GmbH, Wetzlar, Germany). A 63x water-immersion objective (numerical aperture, NA = 1.2) was used to acquire 512 × 512 pixel images of equatorial planes (to avoid photoselection). Calcein was detected by irradiating samples with a 495 nm excitation wavelength and collected at 515 nm, while propidium iodide was recorded with 535 nm excitation light and the emission collected at 617 nm. A femtosecond pulsed titanium-sapphire (Mai-Tai Deepsee, Spectra-Physics) laser tuned at 1000 nm was used for multiphoton simultaneous imaging and IR irradiation of samples. Fluorescence emission was collected by hybrid detectors. Images were analyzed using Leica software.