Cells were seeded at a density of 1.5×105/well in 12-well plates in complete medium. 24hrs after seeding, medium was changed to either fresh complete medium or serum-free medium (SFM). Two days after treatment, cells were harvested with Trypsin, centrifuged and pellets washed once with PBS. Pellets were then re-suspended in 1µg/mL Nile Red in PBS for 5mins and cell suspension analyzed for Nile red fluorescence by flow cytometry using a Beckman Coulter Gallios Machine at Em:488, Ex:580 as previously described [23 (link)].
Gallios machine
Manufactured by Beckman Coulter
Sourced in United States
The Gallios machine is a flow cytometry system designed for analyzing and sorting cells. It provides high-performance data acquisition, analysis, and sorting capabilities for a wide range of applications in life science research.
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Lab products found in correlation
F 7000 fluorescence spectrometer, by Hitachi (2 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Nano zs90 laser particle analyzer, by Malvern Panalytical (1 mentions)
Uv 2600 uv vis spectrometer, by Shimadzu (1 mentions)
Jem 2100 transmission electron microscope, by JEOL (1 mentions)
Fluoview fv500, by Olympus (1 mentions)
Lightcycler480 software setup, by Roche (1 mentions)
Trizol reagent, by Sangon (1 mentions)
Alexa fluor 488 antibody labelling kit, by Thermo Fisher Scientific (1 mentions)
Anti ly6g apc, by BioLegend (1 mentions)
Anti cd11b efluor450, by Thermo Fisher Scientific (1 mentions)
Anti ly6c apc, by BioLegend (1 mentions)
8 protocols using gallios machine
1
Lipid Quantification via Nile Red Staining
2
Fluorescence and Microscopy Techniques
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The fluorescence spectra were measured using a Hitachi F-7000 fluorescence spectrometer (Japan). Confocal laser scanning microscopy (CLSM) studies were performed using an Olympus IX-70 inverted microscope with an Olympus FluoView 500 confocal scanning system. The flow cytometry analysis was performed using a Gallios machine (Beckman Coulter, USA).
3
Cellular Uptake Quantification by Flow Cytometry
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HeLa cells and L02 cells were cultured with the probe (100 nM) in culture media for 3 h. Then the cells were washed with PBS (pH = 7.4) three times and detached from the culture plate by trypsin–EDTA solution. Next, the suspended cell solution was centrifuged at 2000 rpm for 5 min and washed with PBS three times. Finally, the cells were resuspended in PBS for flow cytometric analysis on a Beckman Coulter Gallios machine at 488 nm excitation.
4
Multimodal Nanomaterial Characterization
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All fluorescence measurements were carried out on a F-7000 fluorescence spectrometer (Hitachi, Japan). Ultraviolet-visible light (UV-vis) absorption spectra were recorded on a UV-2600 UV-vis spectrometer (Shimadzu, Japan). The transmission electron microscopy (TEM) images were obtained on a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). Zeta potential and DLS measurements were taken using a Nano ZS90 laser particle analyzer (Malvern Instruments, UK). The confocal laser scanning microscopy (CLSM) images were obtained on a Fluoview FV500 (Olympus, Japan). The flow cytometry analysis was gained from a Gallios machine (Beckman Coulter, USA).
5
Probing HepG2 Cells with Binary Nanosheets
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HepG2 cells (3 × 105) were incubated with binary probes/MnO2 nanosheets for 4 h at 37 °C. Then, the cells were washed three times with PBS and detached with a Trypsin-EDTA solution. Finally, the cells were resuspended in PBS for flow cytometric analysis on a Beckman Coulter Gallios machine.
6
Nanoflare-Mediated miRNA Detection
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LOVE-1, 22Rv1, HeLa and SMMC-7721 cells were incubated with nanoflares. After 6 h, the cells were washed to remove the redundant particles. Cells were then detached from culture dishes using Trypsin-EDTA Solution. The solution containing treated cells was centrifuged (2000 rpm, 4 min) and resuspended in PBS three times. Flow cytometric assay was performed using Beckman Coulter Gallios machine.
Total cellular RNA was extracted from cells using Trizol reagent (Sangon Co. Ltd., Shanghai, China) according to the indicated protocol. The cDNA samples were prepared by using the reverse transcription (RT) reaction with AMV First Strand cDNA Synthesis Kit (BBI, Toronto, Canada). qPCR analysis of miRNAs were performed with SG Fast qPCR Master Mix (2X) (BBI), according to the indicated protocol on an Light Cycler480 Software Setup (Roche). The primers used in this experiment are shown inTable 1 . We evaluated all the data with respect to the miRNA expression by normalizing to the expression of U6 and using the 2-∆∆Ct method.
Total cellular RNA was extracted from cells using Trizol reagent (Sangon Co. Ltd., Shanghai, China) according to the indicated protocol. The cDNA samples were prepared by using the reverse transcription (RT) reaction with AMV First Strand cDNA Synthesis Kit (BBI, Toronto, Canada). qPCR analysis of miRNAs were performed with SG Fast qPCR Master Mix (2X) (BBI), according to the indicated protocol on an Light Cycler480 Software Setup (Roche). The primers used in this experiment are shown in
7
Intracellular Cytokine Profiling of Activated PBMCs
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At day 7 of co-culture, PBMC were harvested and briefly re-stimulated with 50 ng/mL PMA, 0.5 μg/mL ionomycin and 10 μg/mL Brefeldin A, for 4 h at 37°C for intracellular cytokines detection. Dead cells were identified using the LIVE/DEADTM Fixable Dead Cells kit (Invitrogen) following the manufacturer’s instructions. The following anti-human fluorochrome-conjugated Abs were used for surface and intracellular labeling: CD3 (clone SK7), CD4 (SK3), CD25 (M-A251), CD127 (eBioRDR5), Foxp-3 (PCH101), Ki-67 (KI-67), IL-2 (MQ1-17H12), IL-17 (BL168), IFNγ (B27). Abs and corresponding isotype controls were purchased from BD Biosciences, BioLegend and eBioscience. Flow cytometry acquisition was done on a Gallios machine (Beckman Coulter) and analyzed using Flow Jo software.
8
Pristane-Induced Cell Profiling
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Pristane was injected i.p. in WT and CRAMP−/− mice and cells were collected via peritoneal lavage with PBS or after erythrocyte lysis from peripheral blood at day 0 and 7 after injection. Cells were stained extracellularly with anti-CD11b-eFluor450 (eBioscience), anti-Ly6C-APC (BioLegend), anti-CD115-PE (BioLegend), and anti-Ly6G-APC (BioLegend) to determine cell types. Intracellular CRAMP staining was performed with an anti-CRAMP (Innovagen) antibody labelled with an Alexa Fluor 488 antibody labelling kit (LifeTechnologies). Cells were then analyzed by cytofluorometry on a Gallios machine (Beckman Coulter).
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