Following the XTT test, live/dead staining was performed. The modified and unmodified chitosan films as well as the TCPS controls were incubated with 500 μL of DMEM/FBS containing 0.25 μL of 1 mmol/L of Calcein AM (stains live cells green) and l μL of ethD-1 (stains dead cells red) for 25 min at 37 °C. The samples were then imaged with a Nikon Eclipse TS100 fluorescence microscope (Tokyo, Japan) [14 ].
Eclipse ts100 fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Nikon Eclipse TS100 is a fluorescence microscope designed for laboratory use. It is capable of visualizing fluorescently labeled samples, allowing researchers to study cellular and subcellular structures. The microscope features a compact and ergonomic design, making it suitable for a variety of applications in life science research and clinical settings.
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Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (1 mentions)
Sterile pbs, by Thermo Fisher Scientific (1 mentions)
Novared peroxidase substrate, by Vector Laboratories (1 mentions)
In vitro angiogenesis kit, by Thermo Fisher Scientific (1 mentions)
Geltrex matrix, by Thermo Fisher Scientific (1 mentions)
Calcein am dye, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Abnova (1 mentions)
Isolectin b4, by Nikon (1 mentions)
Bio plex multiplex immunoassay kit, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fitc conjugated goat anti rabbit igg, by Boster Bio (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Boster Bio (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Normal melting agarose, by Merck Group (1 mentions)
Ethidium bromide solution, by Merck Group (1 mentions)
Low melting point agarose, by Merck Group (1 mentions)
Annexin 5 fitc pi kit, by Keygen Biotech (1 mentions)
19 protocols using eclipse ts100 fluorescence microscope
1
Live/Dead Cell Viability Assay Protocol
2
Glycolipid-Induced Cell Death Patterns
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To assess the distinct morphological pattern of cell death induced by Glycolipids in HaCaT and SK-MEL-28 melanoma cell line, the cells were stained with acridine orange (AO) and propidium iodide (PI) (Merck, Gillingham, UK) [31 (link)]. HaCaT and SK-MEL-28 cells were cultured, seeded onto Nunc 12-well cell culture plates and treated with glycolipid and synthetic SLES preparations as previously described in Section 2.4 . Following glycolipid treatment, the cells were washed three times with PBS (ThermoFisher Scientific, Loughborough, UK) to remove floating cells and subsequently incubated with AO and PI for 3 min, each prepared at a working concentration of 100 ug mL−1 and mixed at a ratio of 1:1. Cells were then rewashed three times with PBS (ThermoFisher Scientific, Loughborough, UK) and stained cells were imaged at 200× magnification using and Eclipse TS100 fluorescence microscope (Nikon Europe B. V., Amsterdam, The Netherlands). The excitation and emission wavelengths for AO were 493 and 535 nm, and for PI they were 535 and 614 nm. Three images per well were randomly selected (by computer) and processed with ImageJ Software for each of the three independent experimental replicates [32 (link)].
3
Acridine Orange/Propidium Iodide Staining of Treated Cells
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To assess the morphological pattern of cell death induced by mono-RL and Piceatannol treated cells an acridine orange (AO)/propidium iodine (PI) staining technique was utilized [39 (link)]. HCT 116, Caco2 and CCD-841-CoN cells were cultured and treated as previously described in Section 2.7 . Following treatment, cells were washed three times with sterile PBS (Thermo Fisher Scientific) and subsequently incubated for 3 min with 100 μg mL−1 AO and PI (Merck Life Science, Bengaluru, India) mixed at a ratio of 1:1. Cells were then re-washed with sterile PBS (Thermo Fisher Scientific) and stained cells were imaged at 200× magnification using an Eclipse TS100 fluorescence microscope (Nikon). The excitation and emission wavelengths for AO were 493 nm and 535 nm, and for PI were 535 nm and 614 nm. All experiments were carried out in triplicate with three technical replicates per treatment condition. Each replicate was imaged three times and images for publication were randomly selected for publication using a computer. Scale bars (100 μm) were added to images using ImageJ 1.53t software [38 (link)].
4
Evaluating PSMA-Targeted Viral Infection
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J cell derivatives, PSMA-positive (SK-OV-3-PSMA, LLC1-PSMA, Renca-PSMA, LNCaP, PC3-PIP, 22Rv1) and PSMA-negative cancer cells (SK-OV-3, LLC1, Renca, PC3) were infected with R-405 virus at an input multiplicity of infection of 10 PFU/cell as titrated in SK-OV-3-PSMA, for 90 min at 37 °C. Pictures were taken 24 h after infection by Nikon Eclipse TS100 fluorescence microscope. For the neutralization assay, replicate monolayers of the PSMA-positive (SK-OV-3-PSMA, LNCaP, PC3-PIP, 22Rv1) and PSMA-negative cancer cells (SK-OV-3) seeded in 96-well plates were preincubated with increasing amounts of MAb D2B or with mouse IgG for 60 min at 37 °C. R-405 (3 PFU/cell) was added to the MAb-containing medium for additional 90 min. Alternatively, R-405 and R-LM5 virions (3 PFU/cell) were pre-incubated with increasing amounts of HSV-1-neutralizing MAb 52S for 1 h at 37 °C, and then allowed to adsorb to cells for 90 min (R-405 to PSMA-positive cancer cells, R-LM5 to SK-OV-3). Viral inoculum was removed, and cells were overlaid with medium containing MAbs for 18 h. The extent of infection was measured by FACS as the percentage of EGFP-positive infected cells. Western blot assay was performed as described [44 (link)].
5
Immunohistochemical Analysis of SOX2 Expression
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The IHC assays were conducted to detect the expression profile of SOX2 in resected tumors from the sh-NC group and sh-TUG1 group, following the protocols from a previous report (23 (link)). In brief, resected tumors from the xenograft models were fixed in 3% formaldehyde overnight at 4˚C and embedded in paraffin and then cut into 5-µm sections. The slices were then incubated with 0.3% hydrogen peroxide (H2O2) solution in methanol for 20 min at room temperature to block the activity of endogenous peroxidases. Sections were blocked in 10% horse serum at room temperature for 1 h and then incubated with goat anti-SOX2 antibodies (1:100; cat. no. GT15098; Neuromics) at 4˚C overnight, followed by incubation with peroxidase-conjugated horse anti-goat IgG (cat. no. PI-9500-1; 1:2,000; Vector Laboratories, Inc.) at room temperature for 30 min. Immunoreactivity was measured using NovaRed peroxidase substrate (Vector Laboratories, Inc.) under an Eclipse TS100 fluorescence microscope (Nikon Corporation) at x20 magnification.
6
Monitoring Autophagy in Monocytes
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Monocytes (5×105 /well) were cultured in 24-well culture plates. After 24 h of incubation with rWmhsp60 or rapamycin or both, the cells were washed with 2× PBS and incubated with 0.05 mmol/L monodansylcadverine (MDC) (Sigma) at 37°C for 1 h, and the change in fluorescence was observed by Eclipse TS100 fluorescence microscope from Nikon (Tokyo, Japan) at an excitation wave length 380 nm with an emission filter of 525 nm.
7
Angiogenesis Tube Formation Assay
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Tube formation assay was performed using an in vitro angiogenesis kit (Gibco) according to manufacturer's instructions. Briefly, the bottom of the wells of a 48-well plate were coated with 100 µL of reduced growth factor Geltrex matrix (Gibco) and incubated at 37 ˚C for 30 minutes.
HUVECs were then diluted in spent media from control or mechanically stressed ARPE-19 cultures to make a concentration of 1.2510 5 cells/mL. 200 µL of cell suspensions were seeded on Geltrex matrices and incubated for 6 hours at 37 ˚C and in a humidified incubator with 5% CO2 to induce endothelial tube formation. Next, HUVECs were stained with Calcein AM dye (Thermo Fisher Scientific) and imaged using an Eclipse TS100 fluorescence microscope (Nikon Instrument Inc., Melville, NY). Tube length and node numbers were quantified using ImageJ software.
HUVECs were then diluted in spent media from control or mechanically stressed ARPE-19 cultures to make a concentration of 1.2510 5 cells/mL. 200 µL of cell suspensions were seeded on Geltrex matrices and incubated for 6 hours at 37 ˚C and in a humidified incubator with 5% CO2 to induce endothelial tube formation. Next, HUVECs were stained with Calcein AM dye (Thermo Fisher Scientific) and imaged using an Eclipse TS100 fluorescence microscope (Nikon Instrument Inc., Melville, NY). Tube length and node numbers were quantified using ImageJ software.
8
Basophil Identification via Dual Immunostaining
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Sections were simultaneously incubated with a mixture of the primary antibodies against IL-4 (diluted 1:20; Abnova Co., Taipei, Taiwan) and pro-form of major basic protein 1 (proMBP1), followed by their respective secondary antibodies such as donkey anti-rabbit IgG conjugated with Cy3TM (red signal; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and donkey anti-mouse IgG conjugated with Alexa Fluor488 (green signal; Molecular Probes, Eugene, OR, USA) (n = 6). Double-stained sections were observed with a Nikon ECLIPSE TS100 fluorescence microscope. A yellowish signal due to merging of red and green signals was considered to be the result of colocalization of IL-4 in basophils (identified by ProMBP1, a basophil cell marker).
9
Quantification of Immunoreactive Cells
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Immunoreactive cells were counted in each of 10 randomly selected areas of 0.0625 mm2 per slide (i.e., one tissue section) using an objective micrometer under an Olympus light microscope at a magnification of 400 high-power field (HPF). The mean for 10 fields was used as the cell count for that section. After double-immunofluorescence staining, the proportion of cells immunoreactive for ProMBP1 among the cells immunoreactive for IL-4 was determined in 10 randomly selected areas per slide (i.e., one tissue section) at a magnification of 200 HPF with a Nikon ECLIPSE TS100 fluorescence microscope. Data are presented as the median (range).
10
Immunofluorescence Staining of SARS-CoV-2 N Protein
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After infection, the cells were fixed with cold polysorbate for 30 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After washing five times with PBS, cthe ells were incubated with rabbit anti-N antibody (1:500) at 4 °C for 12 h. The cells were then incubated with FITC-conjugated goat anti-rabbit IgG (1:100, KPL, Colton, CA, USA) at 37 °C for 30 min. After washing five times with PBS, the cells were incubated with DAPI (1:1000, diluted in PBS) at room temperature for 20 min. Fluorescence was observed under a Nikon ECLIPSE TS100 fluorescence microscope (Nikon, Tokyo, Japan).
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