Monitoring of Fluorescent Dextran Release from Lysosomes

Why monitor the release of fluorescent dextran from lysosomes

Dextran is a polysaccharide formed by condensation of glucose. The inert and polymeric nature of dextran makes them available in a variety of sizes and easily modified to accept fluorophores. Fluorescent dextran is widely used due to its high water solubility, biocompatibility, and functional range, including nano-drug carriers, cell imaging systems, and nano-biosensors, etc. Lysosomal membrane permeability (LMP) can be monitored by exploiting the cell's stable endocytic capacity, which allows the loading of fluorescent dextran into lysosomes. In healthy cells, fluorescent dextran appears in punctate structures within the lysosome, whereas lysosome-localized dextran translocates into the cytosol upon LMP injury.

Fig. 1. LMP visualized by fluorescent dextran release. (Ellegaard A M, et al., 2015)

Fluorescent Dextran Release from Lysosomes Monitoring Services

Fluorescent dextran formed by dextran-bound fluorescein isothiocyanate (FITC) is commonly used as a macrophage drinking probe to study the properties of endocytic cargoes. CD BioSciences offers multiple methods to quantify and visualize lysosomal permeability during cell death. Here, we visualize LMP by monitoring LMP-induced dextran release into the cytoplasm by fluorescence microscopy.

We developed detailed protocols using fluorescence microscopy to detect individual dextran conjugates whose staining pattern changes from a punctate to a cytoplasmic pattern on the LMP.

1. Seed cells in chamber slides or on glass coverslips and allow to adhere.
2. Add the desired fluorescent dextran to the medium.
3. After dextran loading, wash the cells twice in DPBS and chase for 2 h in fresh medium.
4. Treat the cells for the desired time with an LMP-inducing cytotoxic stimulus.
5. After the proper length of incubation, inspect the cells under a fluorescence microscope.

Characteristics of This Approach

• The extent of LMP can be estimated using dextran of different molecular weights (10-250 kDa): smaller FITC-dextran molecules are released in response to apoptosis-associated LMP, while larger dextran molecules are retained.
• Allows real-time tracking of the LMP process using time-lapse imaging.
• Uses dextran of different sizes and colors to determine the size of the pores formed in the membrane during LMP.
• Can be used to monitor early changes in lysosomal pH during the LMP process.

CD BioSciences can meet any reasonable needs of our clients, taking time and budget into consideration for you. Our aim is to be customer-centric and to provide the highest quality services to customers. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.

Reference

1. Ellegaard A M, Jäättelä M, Nylandsted J. (2015) Visualizing lysosomal membrane permeabilization by fluorescent dextran release[J]. Cold Spring Harbor Protocols. 2015(10): pdb. prot086173.