Cell cycle analysis was performed with BrdU and 7AAD stained siCTRL- or siPATZ1-treated HepG2 cells subjected to flow cytometry. Cell staining with the FITC BrdU Flow kit (BD Biosciences) was performed according to the manufacturer’s instruction. Briefly, HepG2 cells were fixed and permeabilized followed by staining with BrdU and 7AAD. Flow cytometry was performed using the BD LSR Fortessa Flow Cytometry Analyser.
Lsr fortessa flow cytometry analyser
Manufactured by BD
The BD LSR Fortessa Flow Cytometry Analyser is a high-performance instrument designed for multiparameter flow cytometry analysis. It is capable of detecting and measuring various characteristics of cells or particles suspended in a fluid stream.
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5 protocols using lsr fortessa flow cytometry analyser
1
Cell Cycle Analysis of HepG2 Cells
2
CDN Cellular Uptake Mechanism
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CDNcy5.5 as described previously was added to HeLa cells at 37 °C, 5% CO2. The samples were incubated for 6 hours before adding Hoechst 33342 dye and CellMask Orange for nuclei visualization and membrane staining respectively. Samples were washed at least thrice with sterile PBS to remove free CDNs before fixing with 4% paraformaldehyde and were imaged using a confocal microscope (FLUOVIEW FV10i Olympus).
To elucidate if the cellular uptake mechanism of cell derived nanovesicles were via an energy dependent process, CDNcy5.5 were added to HeLa cells and incubated for 4 hours at 37 °C, 20 °C, 4 °C. To further investigate the route of cellular uptake, caveolae inhibitor genistein (200 µM) and clathrin inhibitor chlorpromazine (10 µg/ml) were added respectively to HeLa cells, and incubated for at least 30 minutes before addition of samples under the same conditions. Hoechst 33342 dye was added for live cell visualization. The HeLa cells were subsequently washed three times with sterile PBS, trypsinized and assayed using the BD LSR Fortessa Flow Cytometry Analyser.
To elucidate if the cellular uptake mechanism of cell derived nanovesicles were via an energy dependent process, CDNcy5.5 were added to HeLa cells and incubated for 4 hours at 37 °C, 20 °C, 4 °C. To further investigate the route of cellular uptake, caveolae inhibitor genistein (200 µM) and clathrin inhibitor chlorpromazine (10 µg/ml) were added respectively to HeLa cells, and incubated for at least 30 minutes before addition of samples under the same conditions. Hoechst 33342 dye was added for live cell visualization. The HeLa cells were subsequently washed three times with sterile PBS, trypsinized and assayed using the BD LSR Fortessa Flow Cytometry Analyser.
3
Cell Cycle Analysis via Flow Cytometry
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Cells were transfected with si-Neg or si-Gramd1b for 48 hours, followed by treatment with AG490 for 24 hours. Cells were harvested, and cell pellets were washed in 1X PBS and fixed in 70% ethanol at 4°C overnight. Cell pellets were then washed in 1X PBS and stained with propidium iodide (PI) cocktail containing 50 μg/ml PI (Sigma-Aldrich) and 0.2 mg/ml RNase A (Roche Applied Science). Cells were subsequently subjected to flow cytometry using BD LSR Fortessa Flow Cytometry Analyser, and the percentages of cells in sub-G1 phase were compared using Summit 3.3 software.
4
Characterization of Extracellular Vesicles
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CDNs and exosomes were added to latex beads and incubated for 2 hours at room temperature, to allow physical adsorption of the samples to the beads. The beads were centrifuged at 3000 G for 10 minutes and suspended in PBS thrice to remove free CDNs or exosomes. The primary antibodies CD9, Alix, and TSG101 were added according to the manufacturer’s recommendations and incubated at 4 °C for 1 hour on a shaker, before being centrifuged and resuspended in fresh PBS. Secondary antibodies conjugated to AlexaFluor 488 or AlexaFluor 568 were added and incubated at 4 °C for 1 hour on a shaker. The beads were washed three times by centrifuging at 3000 G for 10 minutes and suspending in PBS each time. Fluorescence was analysed using the BD LSR Fortessa Flow Cytometry Analyser.
5
Mitochondrial Function and Oxidative Stress Assay
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Neurons were detached with Accutase (Sigma, Bornem, Begium) and centrifuged at 300 g for 3 minutes. Batches of 200,000 cells were then incubated in the dye or in the buffer (unstained). MMP was assessed by staining the single‐cell suspension with 200 nM tetramethylrhodamine ethyl ester (TMRE; Invitrogen) for 30 minutes at 37°C. To correct for mitochondrial mass, MitoTracker Green FM (Invitrogen) was used as a counterstaining. For mitochondrial ROS, the single‐cell suspension was stained with 2 μM MitoSOX Red (Invitrogen) for 15 minutes at 37°C without CO2. Cells were analyzed with the BD LSRFortessa flow cytometry analyser and the mean fluorescence intensity of each dye was assessed on at least 20,000 single cells by using FlowJo LLC software. Mean fluorescence of the unstained cells was subtracted to account for autofluorescence.
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