MSCs-derived exosomes were fluorescently labeled using PKH67 dye, which is a green fluorescent dye that labels the lipid membranes. In brief, 100 µg of exosomes was resuspended in 100 µL of diluent C and then mixed with 4 µL of PKH67 dye diluted in 100 µL of diluent C and then incubated for 20 minutes at room temperature; 1 mL of PBS containing 1% BSA was added to stop the labeling reaction and labeled exosomes were reisolated by Exoquick precipitation solution. 4T1 and TUBO cells were cultured in 24-well plate in complete DMEM and when a confluency of 60%–70% was reached, 5 µg of PKH67-labeled exosomes was added to each well and cells were incubated for 24 hours at 37°C with 5% CO2. After incubation, the cells were washed with PBS and fixed in 4% paraformaldehyde for 20 minutes at room temperature. About 0.2 µg/mL of DAPI was added to nuclear staining and then cellular uptake of PKH67-labeled exosomes was visualized using confocal laser scanning microscopy (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany).
Leica tcs spe
Manufactured by Leica Microsystems
Sourced in Germany, United States, Austria
The Leica TCS SPE is a confocal laser scanning microscope. It is designed for high-resolution imaging and analysis of biological samples. The system utilizes advanced optics and a range of laser excitation sources to provide detailed visualization of cellular structures and processes.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Leica application suite, by Leica Microsystems (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Phalloidin atto 488, by Merck Group (2 mentions)
Mitored dye, by Merck Group (2 mentions)
Mitored dye, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Atto 488, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
In situ brdu red dna fragmentation tunel assay kit, by Abcam (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Tunel assay kit brdu red, by Abcam (1 mentions)
Vectashield antifade mounting medium with dap, by Vector Laboratories (1 mentions)
Live dead baclight viability kit, by Thermo Fisher Scientific (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Versa 3d dualbeam, by Thermo Fisher Scientific (1 mentions)
K alphatm, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Uea 1, by Vector Laboratories (1 mentions)
54 protocols using leica tcs spe
1
Fluorescent Labeling of MSC-Derived Exosomes
2
Nanoparticle-mediated Cytotoxicity Assessment
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The potential cytotoxic effect of nHAP/IO and nHAP/IO@miR-21/124 was tested based on ultrastructural features of cells, and mitochondrial metabolism determined using Alamar Blue assay. The metabolic activity of cells measured with Alamar Blue assay was evaluated after 4 days of the experiment, using the protocol established previously and according to the manufacturer's instructions.28 (link) For confocal imaging, cultures were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. The mitochondria network structure was determined after its staining with MitoRed dye (Sigma-Aldrich, Munich, Germany). Actin cytoskeleton was visualised using a solution of phalloidin-Atto 488 (Sigma-Aldrich, Munich, Germany) prepared at a concentration of 1:800 in phosphate-buffered saline pH 7.4. Mitochondria and cytoskeleton staining were described in detail elsewhere.29 (link) The specimens were preserved using Mounting Medium with DAPI ([4ʹ,6-diamidino-2-phenylindole], Thermo Fisher Scientific, Waltham, MA, USA) for nuclei counterstain. The specimens imaging was done with confocal fluorescence microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) under 630× magnification. The analysis was also supported by measuring mRNA-coding anti- and pro-apoptotic molecules, Bcl-2 and Bax, respectively. The description of transcripts determination is described below.
3
TUNEL Assay for DNA Fragmentation
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The TUNEL assay was performed using the TUNEL Assay Kit–In situ BrdU-Red DNA Fragmentation (ab66110; Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. DNA was counterstained with DAPI (Sigma, St Louis, MO, USA) for 10 min 25 °C and washed in PBS. The sections were mounted using the Invitrogen ProLong Gold Antifade reagent with DAPI (Life technologies, Eugene, OR, USA). The images were visualized by confocal microscopy (Leica TCS SPE; Leica microsystems, Wetzlar, Germany) and analyzed using the Leica LAS AF lite 3.2.0 Software.
4
TUNEL Assay for Spermatocyte Apoptosis
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The TUNEL assay was performed using the TUNEL Assay Kit-BrdU-Red (ab66110; Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions but with some modifications according to experimental conditions. Briefly, after treating with primary anti-Sycp3 antibody for 3 h at 4 °C, the spermatocytes were labeled with rTdT reaction mix for 1 h at 37 °C. After washing in PBS, the sections were incubated with anti-BrdU antibody for 30 min at room temperature and washed in PBS. After washing, the slides were incubated with Alexa Fluor 488 Goat Anti-Rabbit Antibody (A11034; ThermoFisher Scientific, Rockford, IL, USA) for 30 min at room temperature. Then, the slides were mounted with VECTASHIELD Antifade Mounting Medium with DAP (Vector Laboratories, Burlingame, CA, USA). The images were visualized by confocal microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) and analyzed using the Leica LAS AF Software lite 3.2.0.
5
Mitochondrial Membrane Potential Assessment
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Changes in MMP were also examined by mitotracker/DAPI florescent dye; Life Technologies (M7512). Briefly, (1.4 μg/mL) of free Cur; (5.5 μg/mL) of free Quer; (1.6 μg/mL) of free Asp; (5 and 10 μg/mL) of CS-SHMP-CQA-NPs [free drugs treatments are chosen as per the drug content in the highest dose of CS-SHMP-CQA-NPs i.e. 10 μg/mL] treated cells were incubated with mitotracker (100 nM) for 15 min at 37 °C and then washed twice with pre-warmed PBS/DMEM. The cells were then fixed with 4% paraformaldehyde for 20 min, permeabilized in triton X-100 (0.1%) for 20 min and stained with DAPI (0.1 µg/mL) for 5 min at 37 °C. After washing the cells with PBS three times, images were acquired by Leica TCS-SPE (Leica Microsystem Nusloch Germany) microscope [65] (link). Fluorescent intensity per cell in four image frames for each group was quantified using NIH ImageJ analysis software (USA) and percent (%) fluorescent intensity (red) was measured by following reported method [20] (link).
6
Quantifying TRAIL Receptor Expression
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Glass slides were placed in 6 well plates and 2 × 105 cells were seeded in each well. Cells were cultured in 2 ml of culture media and incubated for 24 h. After washing with serum-free medium cells were treated with 100 ng/ml or 1,000 ng/ml of TRAIL variants. At the indicated times, the medium was aspirated and the dishes were transferred to ice and washed with cold PBS. Subsequently, cells were fixed in 3% paraformaldehyde for 20 min. In order to analyze the expression of receptors only on the surface of the plasma membrane, the permeabilization step was skipped. After washing with ice-cold PBS and blocking in 3% BSA in PBS for 30 min, the primary antibodies to death receptors DR5 (DR5-01-1) and DR4 (DR-4-02) were added at a concentration of 2 μg/ml and the cells were incubated for 1 h at room temperature. The slides were washed three times with PBS and incubated with 4 μg/ml goat anti-mouse IgG Alexa Fluor 488 and Hoechst 33342 in the dark for 1 h at room temperature. The confocal LSM analysis was performed on Leica TCS SPE (Leica microsystems, Wetzlar, Germany) equipped with immersion ×100 objective with a 1.4 numerical aperture.
7
Visualizing Denture Biofilm Viability
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Biofilms formed on the denture base acrylic resin surfaces were stained using the Live/Dead BacLight Viability kit, comprising SYTO-9 (Invitrogen). Images of the stained biofilms were captured using a CLSM system (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany).
8
Histomorphometric Analysis of Bone-Implant Contact
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After the animals were euthanized, the tibia of the sheep were removed at the specified time periods for histomorphometric analysis. Sections were prepared with nondecalcified histologic slicing system (Exact 300 CL; Exakt Apparatebau, Norderstedt, Germany). The sections were analyzed using a light microscope (Olympus BX60, Tokyo, Japan) to measure the BIC%. All measurements were made using an image analysis software (Olympus Image Analysis System; Olympus Soft Imaging Solutions GmbH, Münster, Germany). The implant surfaces were analyzed in three adjoining microscopic images. The BIC% was measured at a magnification of 40×. The calculation was performed by dividing the length of the attached bone by the length of the complete implant surface (including the whole threads, but the platform surface was excluded). All measurements were made by an independent examiner on two separate days (M.S.T), and the mean values were recorded as final. A confocal scanning laser microscope (CLSM) (Leica TCS SPE; Leica Microsystems, Heidelberg, Germany) was used for fluorescence evaluation.
9
Surface Characterization of Biomaterials
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Surface morphology was examined using a scanning electron microscope (SEM; FEI Versa 3D Dual Beam, Hillsboro, OR, USA), and the surface roughness was quantitatively evaluated using an atomic force microscope (AFM-XE 100 SPM System; Induspia 5F, Suwon, Korea; scan size 50 × 50 μm2). To determine the three-dimensional description of the surfaces (developed interfacial area ratio (Sdr%) and texture aspect ratio (Str); scan size 2 × 2 μm2 with 40×; optical zoom 10×) a confocal laser scanning microscope (CLSM; Leica TCS SPE; Leica Microsystems, Heidelberg, Germany) was used. Energy-dispersive X-ray spectroscopy (EDS; Octane Super SSD, EDAX Corp., Mahwah, NJ, USA) and X-ray photoelectron spectroscopy (XPS) analysis (K-Alphatm; Thermo Scientific, Waltham, MA, USA) were used to decide the chemical configuration of the surfaces.
10
Cytoskeletal Analysis of Colon Cancer Cells
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Fifty thousand HT-29, DLD-1, or SW-480 cells were seeded in 24-well plates with coverslips inserted. The serum starvation and incubation under different oxygen levels with or without JNK inhibitor SP600125 was the same as that of the cell morphological analysis. After incubation, cells were first gently washed by phosphate-buffered saline (PBS) for three times, then fixed with 4% paraformaldehyde (PFA) in PBS for 30 min. After washing by PBS for another three times, the cells were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min. Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells for staining F-actin in 1 h. Cells were mounted with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) after PBS washing. Cells and their cytoskeletons were visualized by confocal microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany). The effect of oxygen level on cytoskeleton was quantified in terms of lamellipodia, filopodia, and ventral stress fibers. At least 200 cells were scored in each of the three independent experiments, and the percentages of cells having the three cytoskeleton structures of different conditions were plotted and compared.
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