Fibroblasts were seeded in round gelatin-coated glass coverslips at a density of 7,400 cells/cm2. To label the lysosomes, fibroblasts were incubated with 70 nM lysoTracker Red DND-99 probe (Invitrogen, Madrid, Spain; λem = 590 nm) for 30 min at 37°C. Next, fibroblasts were fixed with 3% paraformaldehyde (PFA) for 30 min at RT, washed with glycine and stained with 25 μg/ml Filipin (Sigma; λem = 400–484 nm) for free cholesterol detection. Finally, the round covers were mounted with Prolong® Gold reagent (Life Technologies, Alcobendas, Spain) and observed using the fluorescence microscope (Leica, Wetzlar, Germany).
Lysotracker red dnd 99 probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
LysoTracker Red DND-99 probe is a fluorescent dye that selectively stains acidic organelles, such as lysosomes, in living cells. It can be used to visualize and study the distribution and dynamics of these organelles.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Prolong gold reagent, by Thermo Fisher Scientific (1 mentions)
Filipin, by Merck Group (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Tcs sp8, by Leica (1 mentions)
Confocal fluorescence microscope, by Zeiss (1 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
H 1500, by Vector Laboratories (1 mentions)
5 protocols using lysotracker red dnd 99 probe
1
Fibroblast Lysosome and Cholesterol Imaging
2
Visualizing Lysosomes in Live RPE1 Cells
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The LysoTracker staining was performed on live cells as described previously (Chazotte, 2011 (link)). Stable LAMP1‐GFP RPE1 cell lines were generated with the pLVX‐EF1a‐LAMP‐1‐mGFP‐IRES‐Puromycin plasmid (Addgene #134868, a gift from David Andrews) (Schormann et al, 2020 (link)). Then, the cells were grown on coverslips in normal culture medium to ~80% confluence. The LysoTracker Red DND‐99 probe (Invitrogen L7528) was diluted to a working concentration of 50 nM in pre‐warmed (37°C) culture medium and applied to replace the normal culture medium. After the cells were incubated with the probe‐containing medium for 1 h under growth conditions, the loading medium was aspirated, and the cells were washed with fresh normal culture medium to remove any extra dye from the coverslips. The nucleus was stained with Hoechst 33342 (ImmunoChemistry #639) at a concentration of 0.5% v/v. The coverslips were then transferred onto slides and mounted with PBS. The slides were imaged immediately under a confocal microscope (Leica, TCS SP8). The number and size of LysoTracker‐positive puncta per cell were quantified. The colocalization of LysoTraker and LAMP1‐GFP was analyzed using the colocalization tool in Fiji software.
3
Lysosomal Imaging in Fixed Cells
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Cells were plated on coverslips. After treatment, remove the medium from the dish and add the prewarmed LysoTracker™ Red DND-99 probe-containing medium (Invitrogen, United States). Incubate the cells for 1 h at 28°C. Then replace the loading solution with fresh 4% PFA for 15 min. Washed cells two times with DPBS solution and photographed with Zeiss fluorescence confocal microscope at proper fluorescence intensity (577/590 nm).
4
Visualizing Leishmania Phagosome Dynamics
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Macrophages cultivated in the HiQ4 multichamber dishes and infected with DsRed2-expressing L. amazonensis for 24 hours were incubated with 200 nM of Lysotracker Red DND-99 probe (Invitrogen) for a pulse of 30 min, washed and given fresh medium in the microincubator coupled to the confocal unit. The dynamic measurement of PV volumetric enlargement was performed as described [28 (link)], acquiring 10 microscopic fields per experimental condition. PV volumes in μm3 in each analyzed macrophage were graphically represented by a color scale applied to each PV isosurface, ranging from cyan (smaller) to magenta (larger PV). PV volume isosurfaces were also obtained from Dil-ox-LDL fluorescence for correlations between PV size and ox-LDL PV accumulation, using the same methodology. Similar to volume, cell sphericity is a measure obtained from three-dimensional image reconstructions assessed as described [87 (link)].
5
Lysosomal Staining of MiaPaca-2 Cells
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MiaPaca-2 cells were seeded in chamber slides (ThermoFisher, Waltham, MA, USA) and treated with different compounds for 24 h. The next day, cells were washed and stained with LysoTracker Red DND-99 probe (Invitrogen, Carlsbad, CA, USA) in triplicate wells. LysoTracker Red was diluted to a 60 nM concentration and applied for 1 h on live cells. Slides were then mounted with DAPI containing mounting media (Vectashield #H-1500). Slides were visualized using the Zeiss LSM 710 at the UNMC core facility.
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