Reduction in mitochondrial membrane potential (Δψm) is an indicator for mitochondrial and cellular dysfunction [9 (link)]. The Δψm was detected using JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, China). The intensities of red (excitation/emission wave length = 525/590 nm) fluorescence and green (excitation/emission wave length = 490/530 nm) were quantified by the Olympus IX51 inverted microscope system (Olympus, Japan). Then the Δψm was indicated by a decrease in the red/green fluorescence intensity ratio.
Ix51 inverted system microscope
Manufactured by Olympus
Sourced in Japan
The IX51 inverted system microscope from Olympus is a versatile laboratory instrument designed for a wide range of microscopy applications. The core function of the IX51 is to provide high-quality imaging and observation capabilities for various samples and specimens.
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Lab products found in correlation
Ix2 sfr x y stage, by Olympus (3 mentions)
U tvix 2 camera, by Olympus (3 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Lab tek chambered coverglass slides, by Thermo Fisher Scientific (1 mentions)
5 protocols using ix51 inverted system microscope
1
Mitochondrial Membrane Potential Assay
2
Quantifying Apoptosis in Temporal Lobe
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A part of temporal lobes was sliced after being fixed with 4% paraformaldehyde and then it was dehydrated by saccharose phosphate-buffered saline (PBS). TUNEL assay was conducted using the In Situ Cell Death Detection Kit (Roche, USA). The slices were counterstained by 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) and covered by Antifade Mounting Medium (Beyotime, China). Fluorescence microscopy images were observed by the Olympus IX51 inverted microscope system (Olympus, Japan). Apoptosis index (AI) was defined as the mean percentage of TUNEL-positive cells out of six cortical microscopic fields (×400 magnification). The relevant personnel were blind to treatment assignments.
3
Microscopic Analysis of Stained pRBCs
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For microscopy targeting analysis, pRBCs present in the samples were stained by incubation with 0.2 μg/mL Hoechst 33342 (Molecular Probes) and placed into a Nunc™ Lab-Tek™ chambered coverglass prior to image acquisition with an Olympus IX51 inverted system microscope, equipped with an IX2-SFR X-Y stage, a U-TVIX 2 camera, and a fluorescence mirror unit cassette for UV/blue/green excitation and detection of their respective blue/green/red emission ranges.
4
Fluorescent Labeling of Malaria Parasites
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PiRBC suspensions (3% hematocrit, 3% parasitemia) were incubated for 1 h with CF-labeled conjugates 15 or 16 (to a final concentration of 8 µM), at 37 °C. After incubation, cells were washed twice with culture medium and finally re-suspended in a solution of 2 µg/mL of Hoescht 33342 in culture medium, in order to stain the PiRBC nuclei. Cells were then diluted to 0.1% hematocrit in RPMI 1640, and finally deposited onto a Lab-TekTM chambered coverglass slide. Microscopic analysis was performed with an Olympus IX51 inverted system microscope, equipped with an IX2-SFR X-Y stage, a U-TVIX-2 camera, and a fluorescence mirror unit cassette for UV/blue/green excitation and detection of the corresponding blue/green/red emission ranges.
5
Fluorescence Assay for Targeting Efficacy of iLPs
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For fluorescence assays with live cells of the targeting efficiency of iLPs, in vitro RBC suspensions (either blood group B human RBCs or BALB/c mouse RBCs collected in 0.2% w/v EDTA) were brought to 4% hematocrit and washed twice (700 g, 5 min) in RPMI-A before their incubation with one volume of 2× concentrated iLP samples under orbital stirring. Unless otherwise indicated, after 30 min of iLP-RBC incubation, cells were diluted to 0.1% hematocrit in PBS supplemented with 0.75% w/v bovine serum albumin (PBS-BSA) and finally deposited onto Lab-Tek™ chambered coverglass slides (Thermo Fisher Scientific, Inc.). Microscopic analysis was done with an Olympus IX51 inverted system microscope equipped with an IX2-SFR X-Y stage, a U-TVIX-2 camera, and a fluorescence mirror unit cassette for UV/blue/green excitation and detection of the corresponding blue/green/red emission ranges. Phase contrast images were acquired simultaneously. When evaluating iLP performance in vivo in P. yoelii 17XL-infected mice, blood was collected at specific post-administration times in 0.2% w/v EDTA or heparinized tubes when analyzing large (mouse facial vein) or small (terminal portion of animal tail) sample volumes, respectively. pRBC nuclei were stained with 2 µg/ml Hoechst 33342 and cells were finally diluted 1:200 v/v in PBS-BSA prior to fluorescence microscopy analysis as described above.
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