SKOV-3 cells were seeded in a 35 mm Petri dish with a glass cover slide and allowed to adhere overnight at 37 °C under 5% CO2. Cells were then incubated with HAL-2 or HAL-C for 24 h. Cells were stained with the live/dead assay reagents (SYTO-9/PI) and then incubated at 37 °C for 15 min. After washing with 1 mL phosphate buffered saline (PBS), cells were observed by confocal laser scanning microscopy (CLSM; Nikon A1, Japan, 60× oil-immersion objective lens). Green channel for SYTO-9: excitation: 488 nm, emission collected: 500–550 nm; red channel for PI: excitation: 561 nm, emission collected: 570–620 nm (SYTO-9/PI were purchased from Invitrogen).
Syto9 pi
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYTO9/PI is a fluorescent dye system used for nucleic acid staining. It includes the SYTO9 green-fluorescent nucleic acid stain and the propidium iodide red-fluorescent nucleic acid stain. This dye system is commonly used to distinguish between live and dead cells in a sample.
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Lab products found in correlation
Fluorescence microscope, by Zeiss (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Glass bottomed cell culture dishes, by NEST Biotechnology (1 mentions)
Su8010, by Hitachi (1 mentions)
5 protocols using syto9 pi
1
SKOV-3 Live/Dead Cell Imaging
2
Bacterial Viability Assay with Biogenic Silver Nanoparticles
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To judge the viability of bacterial cells upon treatment with the biogenic silver nanoparticles, an SYTO9-PI (Invitrogen, CA) bacterial viability kit was used. An aliquot (10 μl) of a solution consisting of an equal amount of SYTO9 and PI solution was amended into the well of treated cells in each plate and incubated for 15 min in the dark at room temperature. After the incubation, cells were eroded with cold, sterile PBS and fixed on a glass slide. The cells were visualized by fluorescence microscope (Zeiss, USA) (Ahmad et al., 2015 (link)).
3
MRC-3 Fibroblast Viability Assay
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The MRC-3 fibroblasts were cultured in the Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a 5% CO2 atmosphere. In the viability assay, 1 ml of the culture medium containing 2 × 104 of MRC-3 was seeded on the samples (10 × 10 mm) in a 24-well plate. After culturing for 1 and 3 d, the cell viability was evaluated by the MTT assay and SYTO9/PI (Thermo Fisher) staining.
4
AKBA Inhibits Biofilm Formation
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The biofilms were cultured and treated with AKBA as described above in glass-bottomed cell culture dishes (NEST, Wuxi, China). After incubation at 37 °C for 24 h, the dishes were washed with PBS to remove unattached cells. Then, the dishes were stained with 500 µL PBS in the presence of 10 µM SYTO-9/PI (Thermo Fisher Scientific, Waltham, MA, USA) in the dark for 30 min. And the stained biofilms were observed by a CLSM (Zeiss LSM 800, Jena, Germany) with excitation/emission wavelengths of 488 nm/550 nm (SYTO9) and 540 nm/620 nm (PI), respectively (Zhang et al. 2023 (link)).
5
Morphological and Membrane Integrity Analysis of E. coli O157:H7
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SEM was used to observe the morphology of E. coli O157:H7 bacterial cells. The control and treated E. coli O157:H7 bacterial cells were collected by centrifugation (5000 × g, 5 min) and then the bacterial cells were fixed with 2.5 % (v/v) glutaraldehyde for overnight at 4℃. After that, the bacterial cells were washed with 0.01 M phosphate buffer three times and then gradient dehydration with different concentration (30 %, 50 %, 70 %, 80 %, 90 %, 95 % and 100 %) of ethanol. Finally, the samples were critical point drying, sliced, sprayed with gold, and analyzed by SEM (SU8010; Hitachi, Tokyo, Japan) at 10,000 × and 30, 000 ×[28] (link).
The integrity and permeability of E. coli O157:H7 cell membrane were determined by staining with SYTO9/PI (Thermo Fisher Scientific, USA) solution and observed by a CLSM. The E. coli O157:H7 bacterial cells after different treatments were collected by centrifugation (5000 × g, 5 min) and then the bacterial cells were stained with 15 μM of SYTO9/PI solution for 30 min in the dark. After that, the stained bacterial cells were washed with 0.85 % NaCl solution for three times and then 20 μL of stained E. coli O157:H7 cells were dispersed on microscope slide and observed under CLSM [29] (link), [30] (link).
The integrity and permeability of E. coli O157:H7 cell membrane were determined by staining with SYTO9/PI (Thermo Fisher Scientific, USA) solution and observed by a CLSM. The E. coli O157:H7 bacterial cells after different treatments were collected by centrifugation (5000 × g, 5 min) and then the bacterial cells were stained with 15 μM of SYTO9/PI solution for 30 min in the dark. After that, the stained bacterial cells were washed with 0.85 % NaCl solution for three times and then 20 μL of stained E. coli O157:H7 cells were dispersed on microscope slide and observed under CLSM [29] (link), [30] (link).
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