The early versus later stage of apoptosis was analyzed using an annexin V-FITC apoptosis staining/detection kit (Abcam). Wild-type Rp organisms were used to infect AAE2 cells for 4 days, and uninfected cells served as controls. Cells were collected by centrifugation (500 × g for 5 min), resuspended in 1× binding buffer, and incubated with FITC-conjugated annexin V, PI, and NucBlue live cell stain (ReadyProbes reagent; Thermo Fisher Scientific) in the dark for 15 min at room temperature. The cells were then deposited onto microscope slides (Cytospin centrifuge; Thermo Fisher), and imaged on an Olympus BX61 DSU confocal microscope fitted with a 60× objective. Cells were observed using a multiwavelength filter (4′,6-diamidino-2-phenylindole [DAPI], excitation at 365 nm and emission at 480 nm; FITC, excitation at 495 nm and emission at 519 nm; mCherry, excitation at 550 to 590 nm and emission at 550 to 650 nm). All treatments were replicated three times.
Bx61 dsu confocal microscope
Manufactured by Olympus
The BX61 DSU confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a Disk Scanning Unit (DSU) that enables fast and efficient optical sectioning, allowing for the capture of sharp, high-resolution images. The BX61 DSU confocal microscope is a versatile tool suitable for a wide range of research and analysis tasks.
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Lab products found in correlation
Cytospin centrifuge, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc apoptosis staining detection kit, by Abcam (1 mentions)
Fluoroshield mounting medium with dapi, by Vector Laboratories (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Nucblue live cell stain readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using bx61 dsu confocal microscope
1
Apoptosis Analysis by Annexin V-FITC
2
Evaluating Mitochondrial Membrane Potential
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Loss of mitochondrial ΔΨm was detected using a cationic fluorescent redistribution dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1; Immunochemistry) following the manufacturer’s instructions. Host cell-free Rp (MOI = 1) organisms were used to infect cells for 72 h; uninfected cells served as negative controls, and uninfected cells with CCCP served as the positive control. All samples were incubated with JC-1 dye and analyzed for a shift in emission from red (∼590 nm) to green (∼529 nm) using a fluorescence plate reader (Biotek M3). JC-1-stained cells were immobilized onto slides and analyzed under an Olympus BX61 DSU confocal microscope using a 60× objective. Dual fluorescence properties were observed using a multiwavelength filter as described above. All treatments were replicated three times.
3
In Situ Cell Death Detection
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Apoptotic cell death was analyzed using the in situ cell death detection kit (Roche). Cells were immobilized onto slides, fixed in 4% paraformaldehyde for 1 h at room temperature, permeabilized with 0.1% Tween 20 (in phosphate-buffered saline [PBS]), and then incubated with the TUNEL reagents (TdT enzyme-dUTP, 1:10) for 1 h at 37°C. The slides were mounted in Fluoroshield mounting medium with DAPI (Vector Laboratories) and analyzed under an Olympus BX61 DSU confocal microscope with a 60× objective. Dual fluorescence properties were observed using a multiwavelength filter as described above. All treatments were replicated three times.
4
Colocalization Analysis of Transformed Rickettsia
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Cell-free WT and transformed R. monacensis cells were resuspended in complete medium and incubated with NucBlue Live Cell Stain ReadyProbes reagent (1: 50 dilution) (Thermo Fisher Scientific) in the dark for 30 min at room temperature. Fifty-microliter aliquots of cell-free R. monacensis were deposited onto microscope slides (Cytospin centrifuge; Thermo Fisher) at 200 rpm for 3 min. The slides were mounted with 3 μL 1× phosphate-buffered saline (PBS) and imaged on an Olympus BX61 DSU confocal microscope with a 60× objective via a double-wavelength filter (DAPI, excitation at 365 nm and emission at 480 nm; FITC, excitation at 495 nm and emission at 519 nm). Colocalization of fluorescence from gfpuv (transformed R. monacensis) and NucBlue (rickettsial DNA) was analyzed by determining signal overlap for each of three random fields of view using Image Fiji (with the JaCoP plugin and Co-localization Threshold plugin), Pearson’s coefficient (PCC), and calculation of Manders’ colocalization coefficients (MCCs) (26 (link), 27 (link)).
5
Caspase-3/7 Activation Assay in Host Cell-Free Rp
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Caspase enzyme activity was detected using the Magic Red caspase-3/7 assay kit (Immunochemistry), following the manufacturer's instructions. Host cell-free Rp (MOI = 1) organisms were used to infect cells for 72 h, and uninfected cells served as controls. All samples were incubated with Magic Red substrate and then immobilized onto slides. Specimens were mounted in Fluoroshield mounting medium with DAPI and imaged under an Olympus BX61 DSU confocal microscope using a 60× objective. Dual-fluorescence properties were observed using a multiwavelength filter as described above. All treatments were replicated three times.
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