Aliquots of stationary phase cells (400 μl) were pelleted and washed twice in PBS, gently dispersed in 20 μl of PBS, then mixed with 5 μl Nile Red (1 μg/μl) (Sandager et al., 2002 (link); Siloto et al., 2009 (link)). The stained cells were incubated in the dark for 10 min at 30°C, then washed twice in PBS and diluted into 100 μl of PBS. The stained cells were observed and photographed with a fluorescence microscope (Olympus IX71-A12FL/PH, Japan) containing a digital camera. We used Image-Pro plus software (Media Cybernetics, Rockville, MD, United States) to analyze the fluorescence intensity of transgenic yeasts.
Ix71 a12fl ph
Manufactured by Olympus
Sourced in Japan
The IX71-A12FL/PH is an inverted fluorescence microscope system designed for a variety of laboratory applications. It features a 12V 100W halogen illumination system and a motorized focus mechanism. The microscope is equipped with phase contrast capabilities, allowing for the observation of unstained, transparent specimens.
Automatically generated - may contain errors
Lab products found in correlation
Huvecs, by Vector Laboratories (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Huvecs, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
Huvecs, by ScienCell (3 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Transwell plate, by Corning (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Cm dii, by Thermo Fisher Scientific (1 mentions)
4 969 diamidino 2phenylindole dapi, by Vector Laboratories (1 mentions)
8 protocols using ix71 a12fl ph
1
Nile Red Staining for Lipid Quantification
2
Transwell Assay for Cell Migration and Invasion
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Migration and invasion assays were performed using Transwell plates (pore size is 8-µm; Corning, Inc., Corning, NY, USA). In brief, 50,000 cells were seeded into each well with serum-free medium in the upper compartment of the Transwell plates coated with or without BD Matrigel Basement Membrane Matrix (BD Biosciences, Franklin Lakes, NJ, USA), and the ratio of the Matrigel Basement Membrane Matrix and serum-free medium was 1:6. The lower compartment of the chamber was filled with medium with 10% FBS. Following a 24-h culture, the cell numbers that passed through the membrane were fixed with 4% formaldehyde and stained using 0.1% crystal violet and counted under a microscope (IX71-A12FL/PH; Olympus Corp., Tokyo, Japan).
3
Nile Red Staining of Yeast Cells
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Aliquots of stationary phase cells (400 μL) were pelleted and washed twice in PBS, gently dispersed in 20 μL of PBS, then mixed with 5 μL Nile Red (1 μg/μL) [37 ,38 (link)]. The stained cells were incubated in the dark for 10 min at 30 °C, then washed twice in PBS and diluted into 100 μL of PBS. The stained cells were observed and photographed with a fluorescence microscope (Olympus IX71-A12FL/PH, Japan) containing a digital camera. We used Image-Pro plus software (Media Cybernetics, Rockville, MD, USA) to analyze the fluorescence intensity of transgenic yeasts.
4
Quantifying Liver Fat Content
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Rat liver tissues stored at -80 °C were made into frozen sections, stained by the Oil Red O solution (0.5%; Nanjing Jiangcheng Bioengineering Institute, China), observed and photographed by light microscopy (IX71-A12FL/PH; Olympus, Japan). The ratio of red area to total tissue area in each microscopic field was determined by Image Pro Plus 6.0. This ratio represents the relative content of fats in each liver tissue.
5
Exosome-mediated HUVEC Enhancement
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HucMSCs were centrifuged at 100000 g and labeled with the uorescent dye CM-Dil (Moleculai Probes, OR, USA). HUVECs (ScienCell Research Laboratories) at 80% con uence, along with CM-DiI-labeled exos were incubated in Dulbecco's Modi ed Eagle Medium (DMEM). After that, HUVECs were xed in 4% paraformaldehyde, treated with 4',6-diamidino-2-phenylindole (Vector Laboratories, CA, USA) and observed under an inverted uorescence microscope (IX71-A12FL/PH, Olympus, Japan) [22] .
After co-culture with exos containing miR-342-3p agomir, HUVECs were transfected with oe-NC or oe-EDNRA through Lipofectamine 3000 (Invitrogen), and collected 72 h later for subsequent detection.
After co-culture with exos containing miR-342-3p agomir, HUVECs were transfected with oe-NC or oe-EDNRA through Lipofectamine 3000 (Invitrogen), and collected 72 h later for subsequent detection.
6
Exosome-mediated HUVEC Enhancement
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HucMSCs were centrifuged at 100000 g and labeled with the uorescent dye CM-Dil (Moleculai Probes, OR, USA). HUVECs (ScienCell Research Laboratories) at 80% con uence, along with CM-DiI-labeled exos were incubated in Dulbecco's Modi ed Eagle Medium (DMEM). After that, HUVECs were xed in 4% paraformaldehyde, treated with 4',6-diamidino-2-phenylindole (Vector Laboratories, CA, USA) and observed under an inverted uorescence microscope (IX71-A12FL/PH, Olympus, Japan) [22] .
After co-culture with exos containing miR-342-3p agomir, HUVECs were transfected with oe-NC or oe-EDNRA through Lipofectamine 3000 (Invitrogen), and collected 72 h later for subsequent detection.
After co-culture with exos containing miR-342-3p agomir, HUVECs were transfected with oe-NC or oe-EDNRA through Lipofectamine 3000 (Invitrogen), and collected 72 h later for subsequent detection.
7
Exosome-mediated HUVEC Enhancement
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HucMSCs were centrifuged at 100000 g and labeled with the uorescent dye CM-Dil (Moleculai Probes, OR, USA). HUVECs (ScienCell Research Laboratories) at 80% con uence, along with CM-DiI-labeled exos were incubated in Dulbecco's Modi ed Eagle Medium (DMEM). After that, HUVECs were xed in 4% paraformaldehyde, treated with 4',6-diamidino-2-phenylindole (Vector Laboratories, CA, USA) and observed under an inverted uorescence microscope (IX71-A12FL/PH, Olympus, Japan) [22] .
After co-culture with exos containing miR-342-3p agomir, HUVECs were transfected with oe-NC or oe-EDNRA through Lipofectamine 3000 (Invitrogen), and collected 72 h later for subsequent detection.
After co-culture with exos containing miR-342-3p agomir, HUVECs were transfected with oe-NC or oe-EDNRA through Lipofectamine 3000 (Invitrogen), and collected 72 h later for subsequent detection.
8
Fluorescent Labeling of MSC Exosomes
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MSCs were labeled with a fluorescent dye CM-DiI (Molecular Probes, US) by incubating them in the CM-DiI working solution (1 µM) for 15 min at 37°C, followed by washing with PBS and centrifuging at 100,000 g for 2 h at 4°C. The excess dye was removed by precipitation of exosomes. HUVECs were previously cultured to 80% confluency and incubated with DMEM containing CM-DiI-labeled exosomes for 12 h at 37°C with 5% CO 2 . After incubation, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature. The nucleus of cells were stained using medium containing 4,969-diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). Cellular uptake of MSC-derived exosomes by HUVECs was observed using an inverted fluorescent microscope (IX71-A12FL/PH, Olympus, Japan).
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