Quantification of lysosomal pH was determined using Dextran conjugated Lysosensor Yellow/Blue DND-160 (L7545, Invitrogen). Wild-Type and R740 mutant Neruo2a cells were grown in serum starved conditions (DMEM with antibiotics) with 1 µg/ml doxycycline to ∼80% confluence. Cells were then trypsinized, harvested and aliquoted at 100 µl into a black 96-well microplate with 5 µM Lysosensor probe for 15 min at 37°C with 5% CO2 (50 000 cells). The cells were then washed 3× in DMEM and the samples were read in a CLARIOstar Plus spectrophotometer (BMG Labtech) with dual-excitation and dual-emission spectral peaks at 324/441 and 381/541 nm. The ratio of emission was then calculated for each sample as indicated.
Dextran conjugated lysosensor yellow blue dnd 160
Manufactured by Thermo Fisher Scientific
Dextran conjugated Lysosensor Yellow/Blue DND-160 is a fluorescent probe used for the labeling and detection of lysosomes in live cells. It is a ratiometric probe that exhibits a shift in emission wavelength upon changes in lysosomal pH, allowing for the monitoring of lysosomal pH levels.
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2 protocols using dextran conjugated lysosensor yellow blue dnd 160
1
Lysosomal pH Quantification in Neuro2a Cells
2
Lysosomal pH Quantification in Neuro2a Cells
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Quantification of lysosomal pH was determined using Dextran conjugated Lysosensor Yellow/Blue DND-160 (L7545, Invitrogen). Wild-Type and R740 mutant Neruo2a cells were grown in serum starved conditions (DMEM with antibiotics) with 1 µg/mL doxycycline to ~80% confluence. Cells were then trypsinized, harvested and aliquoted at 100 µl into a black 96-well microplate with 5µM Lysosensor probe for 15 min at 37ºC with 5% CO2 (50,000 cells). The cells were then washed 3X in DMEM and the samples were read in a CLARIOstar Plus spectrophotometer (BMG Labtech) with dual-excitation and dual-emission spectral peaks at 324/441 and 381/541 nm. The ratio of emission was then calculated for each sample as indicated.
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