The cellular uptake of the samples was observed by a fluorescent microscope (Eclipse Ti-S, Nikon, Japan). The cells were cultured for 48 h in 24-well flat-bottomed plates, with Mn3O4@PEG-Cy7.5 coated cover glasses in each well. A confocal microscope (TCS SP5 II, Leica, Berlin, Germany) with a laser excitation at 458 and 514 nm was used in the detection. To confirm the endocytosis of the Mn3O4@PEG-Cy7.5, the cellular uptake behavior of the Mn3O4@PEG-Cy7.5 was observed using a confocal laser scanning microscope (CLSM). A549, HEPG2, and PC-3 cells attached to 6-well plates covered with cover glass were treated with Mn3O4@PEG-Cy7.5. After incubation in the dark for a predetermined time, the cells were rinsed twice with PBS (pH 7.4) and fixed using 4% paraformaldehyde solution, and they were then visualized using an inverted fluorescence microscope (Eclipse Ti–S, Nikon, Tokyo, Japan).
Eclipse ti s
Manufactured by Nikon
Sourced in Japan, United States, Germany, China
The Eclipse Ti-S is a high-performance inverted microscope designed for advanced research applications. It features a stable and precise optical system for high-resolution imaging. The Eclipse Ti-S supports a range of observation techniques, including brightfield, phase contrast, and fluorescence microscopy.
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Lab products found in correlation
Coolsnap ez, by Teledyne (12 mentions)
Nis elements software, by Nikon (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
Lsm 710, by Zeiss (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Tcs sp5, by Leica (6 mentions)
Matrigel, by BD (6 mentions)
Nis element, by Nikon (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Sytox green, by Thermo Fisher Scientific (6 mentions)
24 well plate, by Corning (6 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (5 mentions)
Image pro plus, by Media Cybernetics (5 mentions)
Tunel assay kit, by Roche (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Ds fi1c, by Nikon (4 mentions)
Proteinase k, by Thermo Fisher Scientific (4 mentions)
Image pro plus software, by Media Cybernetics (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
628 protocols using eclipse ti s
1
Cellular Uptake of Mn3O4@PEG-Cy7.5 Nanoparticles
2
VSMC Proliferation Assessment via EdU Assay
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We used a 5-Ethynyl-2′-Deoxyuridine (EdU) incorporation assay (C0071S; Beyotime Institute of Biotechnology, Shanghai, China) to detect the proliferation of VSMC. Briefly, VSMC were seeded in 96-well plates. After growing to 60% confluence, the cells were serum-starved for 24 h. After the VSMC were incubated with different concentrations of (R)-TML104 for 4 h and subsequently treated with PDGF-BB for 24 h, and then incubated with EdU for 2 h. Next, the cells were fixed with 4% paraformaldehyde (P0099; Beyotime Institute of Biotechnology, Shanghai, China) for 30 min, permeabilized with 0.1% Triton X-100 for 10 min, and the cells were stained with Hoechst 33342 (50 μL/well) for 10 min. The images were captured using fluorescence microscopy (Nikon Eclipse Ti-S, Tokyo, Japan). The ratio of EdU-positive cells (EdU-stained cells/Hoechst-stained cells ×100%) was determined using a fluorescence microscope (Nikon Eclipse Ti-S, Tokyo, Japan).
3
Fungal Capsule Modulation by MitoDFO
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The yeast form of C. albicans was
seeded in the 2 mL RPMI medium on a 24 well plate with a glass bottom
and treated with MitoDFO in concentrations of 0, 3.6, and 7.2 μM.
The culture was incubated at 35 °C for 24 h, and microscopic
images were taken using phase contrast on an inverted microscope Eclipse
TI-S (Nikon).
C. neoformans capsule
thickness was measured using a previously published protocol.33 (link) Briefly, the yeast was seeded in 1 mL of RPMI
in a 24 well plate with and without a final concentration of 20 μM
mitoDFO for 24 h at 35 °C. The culture was harvested (1300 g,
5 min, RT) and resuspended in 50 μL of RPMI. An equal amount
of India Ink (Thermo-Fisher) was added, and a total volume of 15 μL
of resuspended cells was placed on a microscope slide, covered with
cover glass, and imaged on an Eclipse TI-S (Nikon) inverted microscope
using 100× magnification. A total of 31 cells were randomly selected,
excluding morphologically anomalous and overlapping cells. Using NIS
Elements BR (Nikon), the inner and outer diameters of each cell were
measured, and cell size and capsule thickness were calculated. Graphs
were plotted, and an unpaired t-test was performed
using Prism 8.0 (GraphPad Software).
seeded in the 2 mL RPMI medium on a 24 well plate with a glass bottom
and treated with MitoDFO in concentrations of 0, 3.6, and 7.2 μM.
The culture was incubated at 35 °C for 24 h, and microscopic
images were taken using phase contrast on an inverted microscope Eclipse
TI-S (Nikon).
C. neoformans capsule
thickness was measured using a previously published protocol.33 (link) Briefly, the yeast was seeded in 1 mL of RPMI
in a 24 well plate with and without a final concentration of 20 μM
mitoDFO for 24 h at 35 °C. The culture was harvested (1300 g,
5 min, RT) and resuspended in 50 μL of RPMI. An equal amount
of India Ink (Thermo-Fisher) was added, and a total volume of 15 μL
of resuspended cells was placed on a microscope slide, covered with
cover glass, and imaged on an Eclipse TI-S (Nikon) inverted microscope
using 100× magnification. A total of 31 cells were randomly selected,
excluding morphologically anomalous and overlapping cells. Using NIS
Elements BR (Nikon), the inner and outer diameters of each cell were
measured, and cell size and capsule thickness were calculated. Graphs
were plotted, and an unpaired t-test was performed
using Prism 8.0 (GraphPad Software).
4
Cell Migration and Invasion Assays
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Cell migration assay: 2 × 105 cells were resuspended in 200 μL serum‐free RPMI‐1640 medium and seeded into the upper chamber of each insert. Then, 500 μL medium containing 5% FBS was added to the lower chamber as a chemoattractant. Following incubation at 37°C for 24 hours, the cells on the upper membrane were carefully removed. The cells that migrated were fixed and stained for 30 minutes in a 0.1% crystal violet solution in phosphate‐buffered saline. The membranes were manually counted at ×200 magnification from 10 fields per filter using a Nikon microscope (Nikon Eclipse Ti‐s; Nikon Corporation). All experiments were independently repeated at least three times.
Cell invasion assay: chambers were uniformly covered with 30 μL Matrigel diluted with serum‐free RPMI‐1640 to a certain percentage and incubated at 37°C for 2~4 hours. Then, 1 × 106 cells were resuspended in 200 μL serum‐free RPMI‐1640 medium and seeded into the upper chamber, and 500 μL medium containing 10% FBS was added to the lower chamber. After incubation at 37°C for 24 hours, the cells were fixed and stained. The membrane was manually counted at ×200 magnification from 10 fields per filter using a Nikon microscope (Nikon Eclipse Ti‐s; Nikon Corporation). All experiments were independently repeated at least three times.
Cell invasion assay: chambers were uniformly covered with 30 μL Matrigel diluted with serum‐free RPMI‐1640 to a certain percentage and incubated at 37°C for 2~4 hours. Then, 1 × 106 cells were resuspended in 200 μL serum‐free RPMI‐1640 medium and seeded into the upper chamber, and 500 μL medium containing 10% FBS was added to the lower chamber. After incubation at 37°C for 24 hours, the cells were fixed and stained. The membrane was manually counted at ×200 magnification from 10 fields per filter using a Nikon microscope (Nikon Eclipse Ti‐s; Nikon Corporation). All experiments were independently repeated at least three times.
5
Live/Dead Cell Viability Assay
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Cell viability was assessed using live/dead staining. Cell-laden beads were harvested, rinsed with 0.9% saline, and then incubated in 1 mL of DMEM containing 2 μM calcein-AM (CAM; Sigma) and 2 μM propidium iodide (PI; Sigma) at 37°C for 30 min. Fluorescent images were captured on a fluorescence microscope (Eclipse Ti-S, Nikon). Dead cells appear red and viable cells are green.
6
Nuclei Morphology Visualization with Hoechst 33342
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Hoechst 33,342 (MP, France) staining was performed according to the manufacturer’s instructions. Morphological changes in nuclei were observed under a fluorescence microscope using a blue filter (NIKON ECLIPSE Ti-S).
7
Immunofluorescence Microscopy Protocol
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At 72 hpi, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 10% formalin at room temperature for 20 min. After formalin fixation, the cells were washed three times with PBS and permeabilized with 0.1% Triton X-100 for intracellular staining. The primary antibodies were added at a 1:1,000 dilution in 1% bovine serum albumin in PBS for 1 h. The cells were then washed 3 times with PBS and incubated for 30 min with the secondary antibodies diluted 1:1,000 in 1% bovine serum albumin in PBS. Multiple final washes were done, and the images were taken using a Nikon Eclipse Ti-S.
8
HUVEC Tube Formation Assay
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HUVEC cells were used for the in vitro tube formation assay. Reduced-growth factor Matrigel (Corning, Cat# 354,234) was thawed on ice. Then, 50 μL of Matrigel was added to 96-well plates for polymerization and incubated for 30 min in a 37 °C incubator. A total of 1 × 104 HUVEC cells cultured with the supernatants of the indicated cancer cells were seeded on top of the Matrigel in each well. After incubation at 37 °C for 6 h, images of tube formation in each well were captured using microscopy (Nikon ECLIPSE Ti-s, Japan) and analyzed using ImageJ software.
9
Immunohistochemical Analysis of Tumor Tissues
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Paraffin sections of 4-uM tumor tissues were roasted on Slide Warmer at 60 °C for 1 h, deparaffinized with xylene, and rehydrated with gradient ethanol. The slides were heated with citric acid buffer (pH 6.0) at 95 °C for 20 min for antigen retrieval. The slides were then treated with 3% H2O2 for 10 min to block the endogenous peroxidase reactivity. Subsequently, 5% goat serum was used to block non-specific proteins. The sections were separately incubated with primary antibodies against CD31 (Servicebio, GB11063-1, 1:200), ki67 (Servicebio, GB111141, 1:200), and HIF-1a (Proteintech, 20960-1-AP,1:100). Subsequently, the slides were washed and then incubated with secondary antibodies at room temperature for 30 min. The color was developed with DAB for 1 min and hematoxylin for 1 min. Images were captured using microscopy (Nikon ECLIPSE Ti-s, Japan).
10
CITED1 Gene Silencing in K1 Cells
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Short hairpin RNA (shRNA) targeting the human CITED1 gene and non-targeting shRNA were synthesized by Shanghai R&S Biotechnology Co., Ltd (Shanghai, China). The following RNA interference sequence was transfected into K1 cells to block the expression of CITED1: 5′-TGCTGTATTGGAGATCCCGAGGAACTGTTTTGGCCACTGACTGACAGTTCCTCGATCTCCAATA-3′. K1 cells were seeded in 6-well plates at a concentration of 5×104 cells/well. After K1 cells seeded in 6-well plates were grown to 30% confluence, they were infected with shRNA at multiplicity of infection (MOI) of 50. 5 µL titers of 1×108 TU/mL shRNA were added to each well. Green fluorescent protein (GFP) expression was observed using a fluorescence microscope (Eclipse Ti-S, Nikon, Japan). 72 h after transfection, and the infection efficiency was estimated according to the percentage of green fluorescent chromogenic cells. Followup experiments were conducted when the infection efficiency was above 80%. After that, the transfected cells were evaluated by fluorescence quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting analyses.
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