Jan 11, 2026

Public workspaceMeasurement of Lysosomal pH Using LysoSensor Yellow/Blue DND-160

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7nrx1lwz/v1
  • Ali Ghoochani1,2,3,4,
  • Monther Abu-Remaileh1,2,3,4
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7nrx1lwz/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Measurement of Lysosomal pH Using LysoSensor Yellow/Blue DND-160. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7nrx1lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 08, 2025
Last Modified: January 11, 2026
Protocol Integer ID: 222007
Keywords: measurement of lysosomal ph, quantitative lysosomal ph value, lysosomal ph, ratiometric dye lysosensor yellow, using lysosensor yellow, lysosensor yellow, fluorescence measurement, ph calibration curve, fluorescence, dye incubation
Abstract
This protocol describes the measurement of lysosomal pH using the ratiometric dye LysoSensor Yellow/Blue DND-160. The method includes dye incubation, fluorescence measurement, and the use of a pH calibration curve to derive quantitative lysosomal pH values.
Materials

·       LysoSensor Yellow/Blue DND-160 (Thermo Fisher Scientific)
·       1x DPBS
·       MES calibration buffers (pH 3.5–7.0)
·       eBioscience‱ Monensin Solution (1000X) (Thermo Fisher Scientific, Cat# 00-4505-51)
·     Nigericin sodium salt (Sigma, Cat# SML1779)
·       Microplate reader capable of dual excitation/emission readings
Troubleshooting
Materials
 
LysoSensor Yellow/Blue DND-160 (Thermo Fisher Scientific)
1X DPBS
MES calibration buffers (pH 3.5–7.0)
eBioscience‱ Monensin Solution (1000X) (Thermo Fisher Scientific, Cat# 00-4505-51)
Nigericin sodium salt (Sigma, Cat# SML1779)
Microplate reader capable of dual excitation/emission readings
MES Calibration Buffer Composition:

• 20 mM MES • 20 mM NaCl • 110 mM KCl • 1x monensin • 20 μM nigericin
Procedure:
20m
Culture SH-SY5Y cells in complete media. Once cells become 90% confluence, wash cells with 1XDPBS and add serum-depleted DMEM for 72 hours.
Note
This is a general protocol for measuring lysosomal pH. It can be applied to a variety of cell types, with or without serum stravation depending on the specific experimental conditions.

Wash cells once or twice with 1x DPBS.
Incubate cells (experimental cells and cells for generating pH calibration curve) with 5 μM LysoSensor Yellow/Blue DND-160 in DMEM for Duration00:10:00 at Temperature37 °C

10m
Optional: Wash cells once with 1x DPBS.
Generate a calibration curve by equilibrating LysoSensor-loaded cells in MES calibration buffers with pH values ranging from 3.5 to 7.0.
Incubate cells in each buffer for Duration00:10:00 at Temperature37 °C before measuring fluorescence.

10m
Measure fluorescence using a microplate reader at the following settings:
• Excitation at 340 nm → Emission at 440 nm   
• Excitation at 380 nm → Emission at 540 nm
Calculate the emission ratio (440/540 nm), which correlates with lysosomal pH.
Use the calibration curve to interpolate lysosomal pH from measured emission ratios.