Background of Lysosome Purification by Fluorescence-Activated Organelle Sorting
With the current establishment of large-scale lysosomal histology studies, scientists have developed multiple strategies to isolate intact lysosomes to further understand the function and regulation of the lysosomal pathway. Lysosome purification has become an important tool for lysosomal dysfunction studies. Fluorescence-activated cell sorting is a technique that uses flow cytometry to accurately sort fluorescently labeled individual cells in high-speed linear flow or to qualitatively and quantitatively analyze specific cell populations. Fluorescently labeled organelles can be sorted by this technique for analytical or preparative purposes. Fluorescence- activated organelle sorting (FAOS) has been successfully used to purify lysosomes.
Fig. 1. FAOS was used to isolate secretory lysosomes. (Rajotte D, et al., 2003)
Our Fluorescence-Activated Organelle Sorting Lysosome Purification Services
The principle of FAOS is that fluorescently labeled lysosomes are separated as they pass through the detection laser, thereby separating the fluorescent lysosomes from other organelles. Over the years, CD BioSciences has developed several different lysosome purification techniques. Here, our engineers developed a fluorescence-activated organelle sorting method to purify lysosomes.
Our engineers selected mast cell lines to express fluorescently labeled rat mast cell protease (RMCP-DsRed), which efficiently localizes to secretory lysosomes. Therefore, our FAOS sorting and purification lysosome service is only available for highly purified fluorescently labeled secretory lysosomes from mast cell lines. To obtain a highly purified population of secretory lysosomes, we sorted the lysosomal fraction by the FAOS method.
(1) Autofluorescence analysis was performed on lysosomal fractions from control DsRed cells, and a sorting gate (R1) was established.
(2) The R1 red fluorescent organelle sorting gate was applied to the fluorescent lysosomes present in the lysosomal fraction from cells expressing the RMCP-DsRed fusion protein.
Why Choose Us
- FAOS allows for rapid isolation of lysosomes by flow cytometry.
- FAOS allows to obtain highly purified secreted lysosomal preparations.
- FAOS purified lysosomes show a significant increase in the specific activity of the lysosomal enzyme hexosaminidase.
- FAOS purified lysosomes can be used to build a proteome or for differential protein analysis.
- Large amounts of unclassified material are required for proteomic preparations.
- The main disadvantages of the FAOS technique are that individual lysosomes may break down during sorting and that it is difficult to sort organelles several orders of magnitude smaller than whole cells.
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
- Rajotte D, Stearns C D, Kabcenell A K. (2003) Isolation of mast cell secretory lysosomes using flow cytometry[J]. Cytometry Part A: The Journal of the International Society for Analytical Cytology. 55(2): 94-101.