All microscopy experiments were carried out on a Sp8x confocal laser scanning microscope equipped with a 405 nm diode laser and a pulsed white light laser (Leica microsystems) using 10× Air, 20× Air, and 100× Oil magnification objectives. For the binding assays and the 2D internalization assay, microscope settings were: 50% laser power, 6% shutter intensity for the 405 nm laser line, and 5% for the 594 nm laser line, respectively. Images were acquired consecutively to prevent cross talk between dye excitations or bleeding of emissions in different channels. The scan speed was 200 Hz and the fluorescent signal was visualized using hybrid detectors in BrightR mode, with line accumulation and frame averaging set to three. Settings for the 3D internalization assays were identical, but the shutter intensities were 3% for the 405 nm laser line and 10% for the 594 nm laser line, or 485 nm laser line for doxorubicin excitation. Images were acquired and processed using LasX software, version 1.8.1.13759 (Leica, Wetzlar, Germany).
Sp8x confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany
The SP8X confocal laser scanning microscope is a high-performance imaging system designed for advanced microscopy applications. It features a range of laser excitation sources, sophisticated optics, and a sensitive detection system to provide detailed, high-resolution images of biological and material samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Pulsed white light laser, by Leica (1 mentions)
Mz16f fluorescence stereomicroscope, by Leica (1 mentions)
Dp73 digital camera, by Olympus (1 mentions)
Tritc phalloidin, by Beyotime (1 mentions)
Glass bottom dish, by Corning (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nunc lab tek 2 chamber slide system, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Las x software, by Leica (1 mentions)
Alexa fluor 568 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti cd63, by Thermo Fisher Scientific (1 mentions)
23 protocols using sp8x confocal laser scanning microscope
1
Confocal Microscopy Imaging Protocols
2
Chloroplast Positioning Analysis in Thalli
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The thallus pieces were observed under an MZ16F fluorescence stereomicroscope (Leica Microsystems). To observe chlorophyll autofluorescence of the chloroplasts, a 480/40‐nm excitation filter and LP510‐nm barrier filter were used (Ogasawara et al., 2013 ). RGB digital images (800 × 600 pixels) were captured with a DP73 digital camera (Olympus). Each captured image was analysed with ImageJ software (http://rsb.info.nih.gov/ij/ ) (Rasband 1997–2018). The P/A ratio evaluation method was used to quantify the chloroplast positioning in the cells (Kodama et al., 2008 (link)). The brightness ratio of chlorophyll fluorescence from chloroplasts along the periclinal (P) and anticlinal (A) cell walls was calculated (Kodama et al., 2008 (link)). Fluorescent intensities from 30 points (42 μm each) and 30 areas (196 μm2 each) along the periclinal and anticlinal cell walls were measured. The average P/A ratio with one standard deviation was calculated from three replicated experiments. All statistical analyses in this study were performed using Tukey's HSD test. Furthermore, chlorophyll autofluorescence from chloroplasts from A. endiviifolia and M. polymorpha thalli was observed under an SP8X confocal laser scanning microscope (Leica Microsystems) with excitation at 488 nm and emission at 641 to 721 nm.
3
Cellular Uptake and Imaging of Nanoliposomes
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TR146 cells were seeded in a glass-bottom dish (Corning) at a density of 5 × 104 cells/dish and incubated at 37 °C under 5% CO2 for 24 h to allow the cells to adhere. The culture medium was discarded and replaced with fresh, serum-free medium containing serially diluted nanoliposomes (100 μg/mL, final concentration). The cells were incubated at 37 °C under 5% CO2 for 2 h, washed three times with pre-chilled PBS at 4 °C, and fixed with 4% paraformaldehyde in PBS (w/v) for 20 min at room temperature (RT). The cells were rinsed three times with PBS and permeabilized with 0.5% Triton X-100 for 5 min at RT. The cells were then washed three times with PBS, stained with 500 μL TRITC phalloidin (Beyotime Biotechnology), and incubated for 50 min at RT. After washing three times with PBS, the cells were counterstained with 500 μL DAPI and stored at 4 °C. Fluorescence was observed under an SP8X confocal laser scanning microscope (Leica, Wetzlar, Germany). DAPI (green fluorescence) was measured at excitation and emission wavelengths of 364 and 454 nm. TRITC (red fluorescence) was measured at excitation and emission wavelengths of 545 and 570 nm. FITC-labeled insulin (green fluorescence) was measured at excitation and emission wavelengths of 490 and 525 nm.
4
Imaging S. aureus Iron Uptake
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S. aureus Newman and its isogenic ΔHrtA, ΔHtsA, and ΔIsdD
mutants were grown in either iron-rich or iron-poor medium overnight
for 16–20 h. Then, microbial cultures were diluted to an optical
density of 0.5 MacF units. Cells were incubated with 10 μM Ga3+MPIX for 2 h at 37 °C with shaking. In control cells,
tested compounds were not added. Bacterial samples were washed once
in PBS buffer. Afterward, cells were imaged using a Leica SP8X confocal
laser scanning microscope with a 100× oil immersion lens with
excitation at 405 nm and fluorescence emission at 551–701 nm
(Leica, Germany).
mutants were grown in either iron-rich or iron-poor medium overnight
for 16–20 h. Then, microbial cultures were diluted to an optical
density of 0.5 MacF units. Cells were incubated with 10 μM Ga3+MPIX for 2 h at 37 °C with shaking. In control cells,
tested compounds were not added. Bacterial samples were washed once
in PBS buffer. Afterward, cells were imaged using a Leica SP8X confocal
laser scanning microscope with a 100× oil immersion lens with
excitation at 405 nm and fluorescence emission at 551–701 nm
(Leica, Germany).
5
Subcellular Localization of GmNAC109
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The full-length coding sequence (CDS) of GmNAC109, excluding the stop codon, was amplified with primers containing 25-bp attb sites (Supplementary Table S1
6
Fluorescent Labeling of mRNA-Loaded Nanoparticles
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mRNA was labeled with Cy5 following the manufacturer’s protocol for the Label IT Tracker Intracellular Nucleic Acid Labeling Kit. Labeled mRNA was complexed into four NP-(mRNA)-PPHs in the same manner as unlabeled NP-mRNA-PPH. Furthermore, 4T1, HepG2, and C6 cells were seeded at 10,000, 30,000, and 10,000 cells per well, respectively, in 8-well glass chambers. After 24 h, four labeled NP-mRNA-PPHs were then added to cells at 2 μg/mL mRNA concentration, incubated for 10 h before adding 75 nM of Lysotracker Red DND reagent, and then incubated for another 1 h. All cells were then washed three times with cold PBS and fixed with paraformaldehyde (4% in PBS) for 15 min at room temperature. The fixed cells were further washed with cold PBS three times. NucBlue FixCell ReadyProbe DAPI reagent was diluted 10 times in cold PBS and 100 μL was added to each well. Confocal images were acquired using a Leica SP8X confocal laser scanning microscope (Wetzlar, Germany).
7
Paraformaldehyde-Fixed Cell Immunostaining
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Cells fixed in 4% paraformaldehyde (PFA) were stained with primary and relative secondary antibodies, and imaged on a Leica SP8-X confocal laser scanning microscope.
8
Visualization of NINJ1 in Nigericin-Stimulated BMDMs
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Clone D1 variable domains were cloned into the human Fab expression vector 1AP39.hIgG1.D.Fab (Genentech). Protein was expressed in E. coli and purified with a low endotoxin level (<0.07 EU mg−1). Pam3CSK4-primed BMDMs were cultured with 50 μg ml−1 D1 Fab and then stimulated with nigericin on glass-bottom 96 MicroWell Optical Plates (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde in PBS and then permeabilized with 0.1% Tween-20. Cells were blocked in PBS supplemented with 0.2% fish gelatin (Millipore Sigma), 3% Bovine Serum Albumin and 0.1% Tween-20 for 1 h at room temperature, and then labelled with anti-mouse NINJ1 clone 80 (rabbit IgG2b raised against the N-terminal extracellular domain; Genentech, 2 µg ml−1) at 4 °C overnight. Bound antibody was revealed with an AF488-conjugated anti-rabbit secondary (Thermo Fisher Scientific, 1:200) at room temperature for 1 h. High-resolution images were acquired with a Leica SP8X confocal laser scanning microscope running Leica LAS X v3.5.7 software and equipped with a white light laser and a HC PL APO CS2 oil immersion 40X lens of numerical aperture 1.3.
9
Cellular Uptake of DOX Nanoformulations
10
Labeling and Imaging of NP-mRNA Complexes
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mRNA was labeled with Cy3 following the manufacturer’s protocol for the Label IT Tracker Intracellular Nucleic Acid Labeling Kit. NP (IOSPM, IOCCP, IOCCP-2k PEI, or IOCCP-25k PEI) was tagged with Cy5 by reacting 2 μL (5 mg/mL) NHS-Cy5 with 1 mg/mL NP solution (in 20 mM HEPES buffer, pH 7.4) before 30 min incubation and purification via size exclusion chromatography PD-10 column packed with S-200 resin. Labeled mRNA and NPs were complexed into four NP-(mRNA)-PPHs in the same manner as unlabeled NP-mRNA-PPH. Furthermore, 4T1, HepG2, and C6 cells were seeded at 10,000, 30,000, and 10,000 cells per well, respectively, in 8-well glass chambers. After 24 h, four labeled NP-mRNA-PPHs were then added to cells at 2 μg/mL mRNA concentration, and incubated for 10 h. All cells were then washed three times with cold PBS and fixed with paraformaldehyde (4% in PBS) for 15 min at room temperature. The fixed cells were further washed with cold PBS three times. NucBlue FixCell ReadyProbe DAPI reagent was diluted 10 times in cold PBS and 100 μL was added to each well. Confocal images were acquired using a Leica SP8X confocal laser scanning microscope (Wetzlar, Germany).
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