The distribution and fluorescence intensity of acidic endosomes in the cells were measured with LysoSensor DND-189 dye (Molecular Probes, Eugene, OR, United States) using live-cell imaging (Lusamba Kalonji et al., 2015 (link)). The cells on coverslips in Petri dishes were observed under a fluorescence microscope (Olympus IX70; Olympus Co., Ltd., Tokyo, Japan). The excitation wavelength was 443 nm, and the light emitted from the cells was detected using a 505 nm filter. Fluorescence intensity was calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani Co. Ltd., Fukui, Japan) equipped with a fluorescence microscope. HNE cells were treated with kakkonto (20 μg/ml) or media alone. The fluorescence intensity of the acidic endosomes was measured in 100 cells, and the mean value of the fluorescence intensity was expressed as a percentage of the control value compared to the fluorescence intensity of the cells prior to treatment.
Lysosensor dnd 189 dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
LysoSensor DND-189 dye is a fluorescent probe used for the detection and visualization of acidic organelles, such as lysosomes, in live cells. It selectively accumulates in acidic compartments, emitting fluorescence upon protonation. The dye is membrane-permeant and can be used for real-time monitoring of lysosomal pH and dynamics.
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Lab products found in correlation
6 protocols using lysosensor dnd 189 dye
1
Quantifying Acidic Endosome Fluorescence
2
Endosomal Fluorescence Intensity Measurement
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The distribution and fluorescence intensity of acidic endosomes in the cells were measured with LysoSensor DND-189 dye (Molecular Probes, OR, USA) using live-cell imaging [15 ]. The fluorescence intensity was calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani, Fukui, Japan).
3
High Temperature Effects on Viral Endosomes
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We examined the effects of high temperature on acidic endosomes through which viral RNA enters the cytoplasm [11] (link) to determine the mechanisms by which high temperature alters viral replication. The distribution and fluorescence intensity of the acidic endosomes in cells were measured with LysoSensor DND-189 dye (Molecular Probes, Eugene, OR, USA) and live-cell imaging using previously described methods [39] (link). Cells cultured on coverslips in petri dishes were observed with a fluorescence microscope (OLYMPUS IX70; OLYMPUS Co. Ltd., Tokyo, Japan). The excitation wavelength was 443 nm, and the light emitted from the cells was detected through a 505-nm filter. The fluorescence intensity was calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani Co. Ltd., Fukui, Japan) equipped with a fluorescence microscope. Uninfected cells were cultured on coverslips in petri dishes at 33 °C, 37 °C, 39 °C or 40 °C for 24, 72 or 120 h. The fluorescence intensity of the acidic endosomes was measured in 100 cells, and the mean value of the fluorescence intensity was expressed as a percentage of the control value compared with the fluorescence intensity of the cells cultured at 37 °C.
4
Quantifying Acidic Endosome Fluorescence
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The distribution and fluorescence intensity of acidic endosomes in the cells were measured as previously described using LysoSensor DND‐189 dye (Molecular Probes, Eugene, OR) 31 .
Live‐cell imaging was performed. The cells were cultured on coverslips in Petri dishes and observed using a fluorescence microscope (OLYMPUS IX70; OLYMPUS Co. Ltd., Tokyo, Japan). The excitation wavelength was 443 nm, and the emitted light from the cells was detected using a 505‐nm filter. Fluorescence intensities were calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani Co. Ltd., Fukui, Japan) that was equipped with a fluorescence microscope. The fluorescence intensity of acidic endosomes was measured in 100 HNE cells, and the mean value of the fluorescence intensities was expressed as the percentage of the control value and compared to the fluorescence intensity of the cells that were pretreated with vehicle, as described below.
Live‐cell imaging was performed. The cells were cultured on coverslips in Petri dishes and observed using a fluorescence microscope (OLYMPUS IX70; OLYMPUS Co. Ltd., Tokyo, Japan). The excitation wavelength was 443 nm, and the emitted light from the cells was detected using a 505‐nm filter. Fluorescence intensities were calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani Co. Ltd., Fukui, Japan) that was equipped with a fluorescence microscope. The fluorescence intensity of acidic endosomes was measured in 100 HNE cells, and the mean value of the fluorescence intensities was expressed as the percentage of the control value and compared to the fluorescence intensity of the cells that were pretreated with vehicle, as described below.
5
Quantifying Acidic Endosome Fluorescence
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The distribution and fluorescence intensity of the acidic endosomes in the cells were measured with LysoSensor DND-189 dye (Molecular Probes, Eugene, OR) as previously described (Suzuki et al. 2001a ) using live-cell imaging. Cells on coverslips in Petri dishes were observed with a fluorescence microscope (OLYMPUS IX70; OLYMPUS Co. Ltd., Tokyo, Japan). The excitation wavelength was 443 nm, and the emitted light from the cells was detected through a 505-nm filter. The fluorescence intensity was calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani Co. Ltd., Fukui, Japan) equipped with a fluorescence microscope. HTE and HNE cells were pretreated with EM900 or vehicle or cultured in medium alone. The fluorescence intensity of the acidic endosomes was measured in 100 cells, and the mean value of the fluorescence intensity was expressed as a percentage of the control value compared with the fluorescence intensity of the cells prior to any treatment.
We studied the effects of a long period of pretreatment with EM900 (10 μmol/L, 72 h) on acidic endosomes because the cells were pretreated with EM900 for 72 h prior to RV14 infection.
We studied the effects of a long period of pretreatment with EM900 (10 μmol/L, 72 h) on acidic endosomes because the cells were pretreated with EM900 for 72 h prior to RV14 infection.
6
Acidic Endosomes in RV RNA Entry
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The distribution and fluorescence intensity of acidic endosomes through which RV RNA enters the cytoplasm (Pérez and Carrasco 1993; Casasnovas and Springer 1994) in A549 cells were measured with LysoSensor DND-189 dye (Molecular Probes, Eugene, OR, USA) (Yamaya et al. 2014 ). A549 cells were pretreated with air or 100 ppm CO exposure for 10 min each day for 72 h.
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