The transduced cells were clonally expanded by limiting dilution as described previously 15 (link). Briefly, clones in good condition were picked out two to three weeks later, then, the cells were cultured and passaged once every three days at a ratio of 1:5. The fluorescence from the eGFP was examined under an Olympus Model BX41 fluorescent microscope (Olympus, Tokyo, Japan) and mean fluorescence intensities were measured by a FACS Calibur flow cytometer (Becton Dickinson, MA, USA). The transduced cells were also evaluated for RABV G expression by Western Blot using anti-His monoclonal antibody.
Model bx41 fluorescent microscope
Manufactured by Olympus
Sourced in Japan
The Olympus BX41 is a fluorescent microscope designed for biological and medical research applications. It features a high-intensity illumination system, multiple observation methods, and a robust construction for reliable performance.
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3 protocols using model bx41 fluorescent microscope
1
Clonal Expansion and Characterization of Transduced Cells
2
Lentiviral Transduction of 293T Cells
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A total of 4x104 293T cells /well were prepared in a 24-wells plate. On the following day, the cells in each well were transduced with packaged recombinant lentivirus at an MOI of 1000 (1000 viral genomes per cell) in DMEM medium containing 10% FBS with 6-8 μg /ml hexadimethrine bromide (Polybrene, Sigma, Germany). After 24h, transduction media was replaced with fresh DMEM with 10% FBS and incubated for 3-5 days at 37℃ and 5% CO2. The transduced cells were passaged once every three days at a ratio of 1:10. The fluorescence from the eGFP was examined under an Olympus Model BX41 fluorescent microscope (Olympus, Tokyo, Japan) and provided a marker for evaluating transgene expression of the transduced cells. The transduction efficiency of lentivirus-GFP and mean fluorescence intensities were measured by a FACS Calibur flow cytometer (Becton Dickinson, MA, USA) at 5 and 9 weeks post transduction.
3
Evaluating Mutant AAV-DJ-GFP Vectors
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To assess the efficacy of the mutant vectors we generated, 293 T, Hela and HepG2 cells were either mock infected or infected with 1000 vector genomes (vg)/cell of AAV-DJ-GFP or its K/S/T mutant vectors. At 48 h post-transduction, GFP expression was observed and imaged on an Olympus Model BX41 fluorescent microscope (Olympus, Tokyo, Japan) and the GFP positive efficiency and mean fluorescent signal intensities (MFI) were measured by flow cytometry (FACS Calibur, BD, USA).
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