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LysoFix-GBA (Lysosomal GCase Probe Yellow)

Pricing & Availability
Regulatory Status
RUO
Other Names
FQ10, LysoFix-GBA, GBA substrate, GBA fluorescent substrate, GCase probe
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Product Citations
publications
a. LysoFix-GBA_1_033125
  • a. LysoFix-GBA_1_033125
  • b. LysoFix-GBA_2_033125
    GCase/GBA1 activity detection on the indicated cell lines using LysoFix-GBA (Lysosomal GCase Probe Yellow) live cell imaging (yellow) and Hoechst nuclear staining (blue).
  • c. LysoFix-GBA_3_033125
    Time-course analysis of GCase activity in A549 cells was conducted by incubating the cells with LysoFix-GBA (Lysosomal GCase Probe Yellow) for 2 hours. Following incubation, cells were washed and resuspended in phenol-free media. Live-cell imaging was performed over an 8-hour period, with representative images captured at 0, 4, and 8 hours using a Revvity Operetta CLS™ High Content Analysis System equipped with a 63X objective. Hoechst staining (blue) was used for nuclear visualization.
  • D. LysoFix-GBA_4_051525
    A-549 cells untreated (black line histogram) or treated with the LysoFix-GBA (Lysosomal GCase Probe Yellow) (purple filled histogram) for 2 hours. The cells were fixed using Fixation Buffer (Cat. No. 420801) and analyzed by flow cytometry using a TRITC equivalent channel.
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Description

The enzyme glucocerebrosidase/GCase (encoded by the GBA1 gene) is essential for the hydrolysis of glucocerebroside, a lipid substrate within lysosomes. This enzymatic function is critical for maintaining cellular lipid metabolism and lysosomal integrity. Mutations in the GBA1 gene can result in diminished or defective glucocerebrosidase activity, leading to the intracellular accumulation of glucocerebroside. This accumulation is the primary etiological factor in Gaucher disease, a lysosomal storage disorder manifesting with hepatosplenomegaly, skeletal abnormalities, and neurological complications. Furthermore, GBA1 mutations are recognized as significant genetic risk factors for Parkinson's disease, as they compromise lysosomal function and disrupt cellular homeostasis, thereby contributing to neurodegenerative processes.

This LysoFix-GBA (Lysosomal GCase Probe Yellow) probe, a cell-permeable reporter for GCase activity, fluoresces upon glucocerebrosidase activation, facilitating the detection and monitoring of GBA1 activity. This is pivotal for the diagnosis and management of related diseases, enabling early diagnosis, elucidation of disease progression, and the development of targeted therapies. Thus, comprehending the function and activity of GBA1 is imperative for advancing therapeutic strategies and improving disease management for these conditions. Additionally, a correlation between GCase activity in iPSC-derived neurons and monocytes suggests that patient blood samples may serve as a surrogate for GCase activity in patient neurons.

Product Details
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Human
Formulation
Lyophilized
Preparation
See application notes.
Storage & Handling
-70°C
Application

ICC, Live cell imaging, FC - Verified
Recommended Usage

FC, ICC & Live cell staining: See Application Notes

Application Notes

Component: 
500 µg of LysoFix-GBA (Lysosomal GCase Probe Yellow)

General Considerations: 

  • Optimal concentration and loading time will vary depending on cell type, application and experimental conditions. Recommended working concentrations range between 2.5 -20 μM LysoFix-GBA (Lysosomal GCase Probe Yellow).
  • LysoFix-GBA (Lysosomal GCase Probe Yellow) is compatible with kinetic imaging and analysis for up to 8 hours in cell culture and under appropriate conditions. 
  • AT3375 and isofagomine are potent Glucocerebrosidase inhibitors that can be used for control samples. An effective AT3375 and isofagomine concentration is typically 10 μM for overnight incubation at 37°C prior to LysoFix-GBA (Lysosomal GCase Probe Yellow) addition for most cell types. We recommend users to determine optimal conditions based on their assay.

Reconstitution and Preparation of Stock Solution:

  1. Thaw and bring LysoFix-GBA (Lysosomal GCase Probe Yellow) to room temperature in dark for at least 10 mins
  2. Add 45.9 µL of DMSO to make a stock solution of 5 mM. Vortex (maximum speed) for 60s and briefly centrifuge.
  3. Aliquot the stock immediately into several one-time-use tubes of desired volumes. Avoid freeze thaw cycles with this material and always keep in the dark.
  4. We do not recommend re-freezing any unused stock solution after a single aliquot has been thawed for experimental use.

Preparation of Working Solution:

  1. Allow the aliquot of LysoFix-GBA (Lysosomal GCase Probe Yellow) stock solution to thaw and reach room temperature protected from light.
  2. Determine the desired working concentration and volume of LysoFix-GBA (Lysosomal GCase Probe Yellow) for your assay.
  3. Prepare the working solution (between 2.5 -20 μM) by diluting the LysoFix-GBA (Lysosomal GCase Probe Yellow) stock solution with the desired assay buffer (such as phenol red-free culture media or PBS).

Microscopy assay (live cell imaging and ICC)

This protocol is designed for adherent cell lines. It is recommended to use surface coating, such as poly-L-Lysine, to promote cell adhesion and avoid washing off cells during wash steps. If working will cells in suspension, optimal assay conditions may need to be determined.

  1. Gently remove media from cells which were seeded onto the desired live cell imaging plates. 
  2. Gently add the working solution of LysoFix-GBA (Lysosomal GCase Probe Yellow). Incubate at 37°C for 2 hours and protected from light.
  3. Wash wells with an excess of phenol red-free medium or buffer of choice. Repeat wash 2 times for a total of 3 washes. Be careful not to disrupt cell adhesion.
  4. Add the desired amount of phenol red-free medium supplemented with 2-10% FBS.
  5. Perform live cell imaging using the TRITC channel for LysoFix-GBA (Lysosomal GCase Probe Yellow) detection.

Flow cytometry assay

  1. Prepare a 1X working solution of LysoFix-GBA (Lysosomal GCase Probe Yellow) by diluting the stock solution to 5 µM in the desired phenol red-free media or buffer containing 5% FBS.
    Note: The working concentration of 5 µM for 2 hours was optimized for human PBMCs and CD14+ monocytes. It may be necessary to determine the optimal concentrations for the desired assay conditions and cell types.
  2. For adherent cells, gently remove culture media and replace with 1X working solution of LysoFix-GBA (Lysosomal GCase Probe Yellow) and incubate for at least 2 hours at 37°C and 5% CO2.
    Note: multiple time points may be desired depending on your experimental design.
  3. Then, gently remove the solution from cells and replace with phenol red-free media containing 5% FBS.
  4. Harvest the cells following desired detachment reagent (such as trypsin) and resuspend in the desired flow cytometry buffer/solution.
    Note: Cells can be fixed with PFA 2% or Fixation Buffer (Cat. No. 420801) for 10 min.
  5. Analyze the cells using a flow cytometer using a TRITC or equivalent channel for detection of LysoFix-GBA (Lysosomal GCase Probe Yellow).

Detection/Imaging Guidelines
- Ex/Em: 541/567
- Fluorescence microscope filter set: TRITC
- Flow Cytometer Channel: TRITC or equivalent

Additional Product Notes

This product is intended for single use after reconstitution and performance is affected by frozen-thaw cycles. If more than single use is needed, we recommend store reconstituted product in single use aliquots at -70°C (Please see application notes for details). 

Application References

(PubMed link indicates BioLegend citation)
  1. Deen MC, et al. 2022. Proc Natl Acad Sci U S A.119:29.

Antigen Details

Structure
Chemical Formula: C108H148N26O23
Molecular weight: 2178.53 g/mol
Antigen References
  1. Deen MC, et al. 2022. Proc Natl Acad Sci U S A.119:29.
  2. Gehrlein A, et al. 2023. Nature Communications. 14:2057
  3. Zhu S, et al. 2023. Angew Chem Int Ed Engl. 62,e202309306.
  4. Williams D, et al. 2024. Proc Natl Acad Sci U S A.
  5. Koros C, et al. 2024. Genes (Basel). 15:1605.
Gene ID
NA
Go To Top Version: 1    Revision Date: 03/31/2025

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

BioLegend, the BioLegend logo, and all other trademarks are property of BioLegend, Inc. or their respective owners, and all rights are reserved.

 

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Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

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