Transfection was carried out in 6-well plate format. Plasmid complex, at a total volume of 20 μL/cm2, consist of 250 ng/cm2 DNA, 1.1 µL/cm2 Polyethylenimine MAX (Polyscience; 1 mg/mL) and DPBS, was incubated at room temperature for 15 min. The plasmid complex was then added dropwise into MSCs (150000 cells/cm2 in 6-well plates) supplemented with 500 ng/mL Lipofectamine™ 2000 Transfection Reagent (ThermoFisher Scientific) and 0.5 μM Vorinostat (Histone deacetylase inhibitor; HDACi, BioVision) in complete medium. The culture media was replaced with fresh media at 24 h post-transfection. Then, cells were further incubated for 24 h before analysis. Cell images were taken with EVOS FL Cell Imaging System (Thermo Fisher Scientific) equipped with a GFP (Ex470/Em510) fluorescent light cube. For flow cytometric analyses, cells were washed by DPBS, trypsinized using TrypLE Express. Percentage of fluorescent positive cells was quantified by Attune NxT Flow Cytometer system (ThermoFisher Scientific), and the raw data was analysed using non-modified MSCs as the negative control at < 0.8%, using Invitrogen Attune NxT software (Version 3.1.2, ThermoFisher Scientific).
Attune nxt flow cytometer system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Attune NxT Flow Cytometer system is a high-performance instrument designed for multi-parameter analysis of cells and other biological particles. It utilizes flow cytometry technology to provide rapid and accurate measurements of various cellular properties, including size, granularity, and fluorescence intensity.
Automatically generated - may contain errors
Lab products found in correlation
Attune nxt software, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Hdaci, by Abcam (1 mentions)
Vorinostat, by Abcam (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine max, by Polysciences (1 mentions)
Apc anti mouse cd11b, by BioLegend (1 mentions)
A1r si high speed spectral laser scanning confocal inverted microscope, by Nikon (1 mentions)
9 protocols using attune nxt flow cytometer system
1
Efficient Transfection of Mesenchymal Stem Cells
2
Quantifying Fluorescent MSC Populations
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Cells were washed twice with 1XPBS and trypsinized for 5 min. After detachment of cells, complete media were added at 4 times of the volume of trypsin. The suspension cells were subsequently transferred to conical or Eppendorf tubes. Cell pellets were obtained from centrifugation at 300g for 3 min. Cells were then resuspended in PBS prior to analysis. Percentage of fluorescence positive MSCs was quantified by Attune NxT Flow Cytometer system (ThermoFisher Scientific), and the raw data was analysed using non-modified MSCs as negative controls at < 0.5%, using Invitrogen Attune NxT software (ThermoFisher Scientific).
3
MSC-Mediated Cancer Cell Apoptosis
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CDUPRT_MSC or CDUPRT-IFNb_MSC were co-cultured with ES2 at a ratio of 1 MSC to 1 cancer cell. Two days after the addition of 150 μg/mL 5FC, all cells were harvested and stained with TMRE (biotium) or annexin-V (PE Annexin V Apoptosis Detection Kit with 7-AAD, Tonbo Biosciences) according to the manufacturer’s instructions. Percentage of fluorescent positive cells was quantified by Attune NxT Flow Cytometer system (ThermoFisher Scientific), and the raw data was analysed using Invitrogen Attune NxT software (Version 3.1.2, ThermoFisher Scientific).
4
Murine PBMC Activation by Rickettsial Factors
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Mouse PBMCs (4 × 105 cells) were incubated with culture supernatants of ΔTRP120 or WT-infected or uninfected DH82 cells for 3, 6, and 9 h at 37°C. Harvested PBMCs were resuspended in 100 µL flow cytometry buffer and incubated with anti-mouse CD16/32 Fc blocker (BioLegend), then with APC anti-mouse CD11b (BioLegend) or FITC anti-mouse Ly6C antibodies (BioLegend). Stained cells were fixed and subjected to flow cytometry using the Attune NxT Flow Cytometer System (Thermo Fisher). Data were analyzed in FlowJo software (Ashland, OR).
5
Cellular Uptake of FITC-Labeled PIC
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For flow cytometry studies, B78 cells and RAW264.7 cells were seeded and cultured in 96-well plates with 10,000 cells per well 24 h before treatments. Cells were treated with FITC-labeled PIC at different concentrations (i.e., 1.9, 3.8 and 7.5 μg/mL). Two hours later, the cells were collected by trypsinization and centrifugation. The cellular uptake of FITC-labeled PIC was measured by flow cytometry (Attune NxT flow cytometer system, ThermoFisher) quantifying FITC-positive cells.
For confocal laser scanning microscopy, B78 cells and RAW264.7 cells were seeded on coverslips in 6-well plates and incubated for 24 h. Cells were treated with FITC-labeled PIC at 3.8 μg/mL. After 2 h of incubation, cells were washed with PBS and fixed with 4% paraformaldehyde. Thereafter, cells were stained by 4'−6-diamidino-2-phenylindole (DAPI, 1 mg/mL, 1 μL/well) and washed with PBS. The coverslips were carefully taken out from the wells, placed on slides and enclosed with anti-fade mounting medium. The samples were imaged with a Nikon A1R-Si high speed spectral laser scanning confocal inverted microscope (Nikon, Melville).
For confocal laser scanning microscopy, B78 cells and RAW264.7 cells were seeded on coverslips in 6-well plates and incubated for 24 h. Cells were treated with FITC-labeled PIC at 3.8 μg/mL. After 2 h of incubation, cells were washed with PBS and fixed with 4% paraformaldehyde. Thereafter, cells were stained by 4'−6-diamidino-2-phenylindole (DAPI, 1 mg/mL, 1 μL/well) and washed with PBS. The coverslips were carefully taken out from the wells, placed on slides and enclosed with anti-fade mounting medium. The samples were imaged with a Nikon A1R-Si high speed spectral laser scanning confocal inverted microscope (Nikon, Melville).
6
Intracellular ROS Quantification Using DCFH-DA
7
Flow Cytometric Analysis of Cells
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For flow cytometry analysis (FACS), the cells were trypsinised, centrifuged and re-suspended in PBS. Cell clumps were removed by filtering through 40 mm mesh. Percentage of fluorescence positive cells was quantified by Attune NxT Flow Cytometer system (ThermoFisher Scientific) and the raw data was analysed using Invitrogen Attune NxT software (ThermoFisher Scientific). At least 10,000 cells were analysed per sample.
8
Cellular Uptake of DOX-Loaded SMOF Nanoparticles
9
Oxidative Stress Assessment of Nanoparticles
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