The morphologies of rMSCs cells cultured on PLA and mHA1/PLA films were observed under CLSM one day after inoculation. Before observation, the samples were washed with PBS and fixed with 4% PFA at room temperature for 30 min. After fixation, the cells were infiltrated by 0.1% Triton X-100 for 10 min and blocked with 1% BSA for 30 min; then, each sample was stained with Phalloidin-FITC for the F-actin filaments for 60 min and DAPI for the nucleus for 10 min in the dark. The samples were sealed on microscope slides and observed under a FV3000 CLSM (Olympus, Hitachi, Japan).
Fv3000 clsm
Manufactured by Olympus
Sourced in Japan
The FV3000 CLSM is a confocal laser scanning microscope designed for advanced imaging applications. It features a modular design, allowing for configuration to suit various experimental requirements. The system employs laser excitation and a sensitive detector to capture high-resolution, three-dimensional images of samples.
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Lab products found in correlation
Glass bottom cell culture dish, by NEST Biotechnology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Illustrator cs6, by Adobe (2 mentions)
γ h2aχ mouse monoclonal primary antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Bioss Antibodies (1 mentions)
Picogreen dsdna quantitation reagent, by Yeasen (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Es500w camera, by JEOL (1 mentions)
Jem 1011, by JEOL (1 mentions)
Baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Fluoromax 4, by Horiba (1 mentions)
10 protocols using fv3000 clsm
1
Morphological Analysis of rMSCs on PLA and mHA/PLA
2
Calreticulin Redistribution by Arad-NWs
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CT26 cells were seeded on glass bottom cell culture dish (NEST, 20 mm) with a density of 2 × 105 cells per dish. After 24 h, Vehicle or aAGd-NWs ([Gd] = 50 μM, [ara-AMP] = 50 μM) were added into culture medium and incubated for 24 h followed by X-ray irradiation (0 or 5 Gy × 1). After 4 hours incubation, cells were washed with PBS for three times and stained with CRT-antibody (diluted 1:500 with 1% BSA, Abcam Rb mAb to Calreticulin [EPR3924], UK) for 1 h. Subsequently, tumor cells were washed with PBS for three times and stained with Alexa Fluor 647 conjugated goat anti-rabbit IgG (H&L, diluted 1:200 with 1% BSA, Zen-Bioscience, China) for another 1 h at 37 °C. Then, tumor cells were stained with DAPI (Beyotime, China). Immunofluorescence images were obtained from Olympus FV3000 CLSM and analyzed with ImageJ Software.
3
Intracellular Localization of Gadolinium-based Nanoprobes
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CT26 tumor cells were seeded in a glass bottom cell culture dish (NEST, 20 mm) at a density of 2 × 105 cells per dish. The cells were incubated with ICG@aAGd-NWs ([Gd] = 50 μM, [ara-AMP] = 50 μM; Ex = 789 nm, Em = 813 nm) for 6 h. LysoTracker Green (Ex = 504 nm, Em = 511 nm) and tumor cells were co-incubated for 30 min at 37 °C, washed twice with PBS, and then stained with DAPI (Ex = 364 nm, Em = 454 nm). Then, tumor cells were washed 3 times with PBS and observed via Olympus FV3000 CLSM. Turning on different lasers in CLSM sequentially and separately to prevent crosstalk. Then, ImageJ (Colocalization Finder) was used for the calculation of the Pearson colocalization coefficient (PCC).
4
Gd-Based Nanoparticle Radiosensitization in CT26 Cells
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CT26 cells were seeded in confocal dishes at 2 × 105 per dish and incubated overnight for attachment, which were divided into six groups of Vehicle, Vehicle+RT, Gd-NCPs, GGd-NCPs+RT, aAGd-NWs, aAGd-NWs+RT ([Gd] = 50 μM, [ara-AMP] = 50 μM). After incubation for 6 h, the cells were treated with radiation (0 Gy or 5 Gy × 1). Two hours after irradiation, all treatments were removed, and tumor cells were fixed with 4% paraformaldehyde and washed with PBS. Then, 0.3% Triton-X was used to perforate the nucleus. Then, cells were added with 1% bovine serum albumin solution as a blocking buffer, and incubated with γ-H2Aχ mouse monoclonal primary antibody (1:500, Abcam, UK) for 1 h and washed with PBS. After that, Alexa Fluor 488 conjugated secondary antibody (1:500, Bioss, China) was added and incubated for 1 h. Then, DAPI was used to stain the nucleus. Tumor cells were observed under Olympus FV3000 CLSM, and analyzed by ImageJ software.
5
Cytosolic DNA Damage Detection
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To detect cytosolic DNA damages, CT26 cells were seeded in a glass bottom cell culture dish (NEST, 20 mm) at a density of 2 × 105 cells per dish. The cells were treated with Vehicle, GGd-NCPs, aAGd-NWs with or without X-ray irradiation ([Gd] = 50 μM, [ara-AMP] = 50 μM) and incubated for 24 h. Then, PicoGreen staining was performed 6 h and 24 h post irradiation, respectively. For PicoGreen staining, cells were incubated with PicoGreen dsDNA Quantitation Reagent (diluted 1:200 with PBS, Yeasen, CHINA) for 10 min at 37 °C, washed with PBS and stained with DAPI (Beyotime, China). Fluorescence images were obtained from Olympus FV3000 CLSM and analyzed with ImageJ Software.
6
3D Tumor Spheroid Penetration and Cytotoxicity Assay
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To evaluate the 3D Spheroid penetration, CT26 cells (3 × 103 per well) were seeded into ultralow attachment 96-well plates (Corning, 7007, USA). Indocyanine green (ICG) was integrated into GGd-NCPs and aAGd-NWs to obtain ICG@GGd-NCPs and ICG@aAGd-NWs, respectively. Then, CT26 3D tumor spheroids were co-incubated with ICG@GGd-NCPs and ICG@aAGd-NWs for 8 h. Then the red fluorescence of ICG within 3D spheroids was recorded by Olympus FV3000 CLSM. To delve deeper into the penetration capabilities of ultrafine aAGd-NWs in 3D CT26 spheroids, these spheroids were dissociated into single cells for flow cytometry analysis. The dissociated single-cell suspension was subjected to three washes with PBS and was then collected by short-duration and low-speed centrifugation (2000g × 5 min) to remove unuptaked ICG@aAGd-NWs. Ultimately, the cells treated with ICG@aAGd-NWs were resuspended in PBS for flow cytometry analysis. To test the cytotoxicity of aAGd-NW-sensitized radiation within CT26 3D tumor spheroids, CT26 3D spheroids were incubated with Vehicle, GGd-NCPs ([Gd] = 50 μM), aAGd-NWs ([Gd] = 50 μM, [ara-AMP] = 50 μM) for 12 h and then irradiated (0 or 5 Gy × 1). After 24 h, calcein-AM and propidium iodide (PI) were used to stain live and dead cells and the sizes of 3D tumor spheroids after various treatments were measured by Nikon Eclipse Ti (Japan) at days 5 and 10, respectively.
7
Confocal and Electron Microscopy Imaging
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Samples for confocal laser scanning microscopy (antibody staining and in situ hybridization) were mounted in Murray’s clear and scanned in either Leica SP5 or Olympus FV3000 CLSM. Z-stacks of confocal scans were projected into 2D images in IMARIS 9.1.2. TEM microphotographs were obtained with Gatan ES500W camera mounted on transmission electron microscope Jeol JEM-1011. Both CLSM images and TEM micrographs were assembled in Adobe Illustrator CS6 into final figures. All the schematic drawings were done with Adobe Illustrator CS6.
8
Bacterial Viability Imaging Protocol
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Samples were taken from the zone of inhibition and also from a growth area as a positive control and then smeared on glass slides. The slides were stained using a Baclight™ Bacterial Viability Kit (Molecular Probes, ThermoFisher). Basically, equal volumes of the dye mixture (3.34 mM SYTO/20 mM propidium iodide) were mixed together, and 2 μl was added per ml of phosphate buffer saline pH 7.4. The solution was added onto the glass slides and incubated for 20 minutes. Slides were gently washed, mounted and then imaged using an Olympus FV3000 CLSM at ×60 magnification. The excitation/emission maxima for STYO9 and propidium iodide were 480/500 nm and 490/635 nm. Green or red fluorescence indicates live or dead/compromised cells respectively.
9
Fluorescent Quenching of BSA by SLNs
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The ACQ probe P4 was involved in the CLSM tests to monitor the SLNs. The P4-labeled SLN were incubated with FITC-labeled BSA for 24 h. Then, the mixtures were imaged by FV3000 CLSM (Olympus, Tokyo, Japan) with excitation wavelength set at 488 nm and 640 nm and magnification set at 100. The image display and analysis were performed using the software Olympus FV31S.
With the intrinsic fluorescent property of BSA, the fluorescent quenching of BSA when incubated with SLNs at different concentrations for 0, 24 and 48 h were measured by Fluoromax-4 (HORIBA, USA) using a 3-cm quartz cuvette. The BSA concentration was set at 12 μmol/L, and the concentration range of SLNs was 0–2.5 mmol/L. Excitation was performed at 280 nm with a slit width of 3 nm and the emission was performed from 300 nm to 450 nm with a slit width of 3 nm. The measurement was conducted at pH 7.4.
With the intrinsic fluorescent property of BSA, the fluorescent quenching of BSA when incubated with SLNs at different concentrations for 0, 24 and 48 h were measured by Fluoromax-4 (HORIBA, USA) using a 3-cm quartz cuvette. The BSA concentration was set at 12 μmol/L, and the concentration range of SLNs was 0–2.5 mmol/L. Excitation was performed at 280 nm with a slit width of 3 nm and the emission was performed from 300 nm to 450 nm with a slit width of 3 nm. The measurement was conducted at pH 7.4.
10
Intracellular ROS Quantification in CT26 Cells
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CT26 cells were seeded in a 96-well plate at a density of 8000 cells per well and attached overnight. The cells were incubated with Vehicle, GGd-NCPs, aAGd-NWs ([Gd] = 50 μM, [ara-AMP] = 50 μM) for 6 h. Before X-ray irradiation, H2DCFDA (1:1000, used as ROS probe to detect intracellular ROS generation) was incubated for 1 h at 37 °C and washed with PBS for three times. After irradiation (0 Gy or 5 Gy × 1), DAPI was used to stain the nucleus. Subsequently, immunofluorescence images were obtained from Olympus FV3000 CLSM and analyzed by ImageJ Software.
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