By Alex Pinggera, Senior Scientist II, Metrion Biosciences
To enable the study of ion channels found on the lysosomal membrane we perform the lysosomal patch-clamp technique:
- Cells are seeded out at a high density onto Poly-D-Lysine coated coverslips. This ensures that the cells are well adhered to the coverslip, and that they form a vast network, which facilitates easier slicing and extraction of the lysosomes from the cells.
- The cells are treated overnight with a small chemical called vacuolin-1. This chemical helps to enlarge the lysosomes, so they are big enough to be patched with a pipette.
- The following day, the coverslip is incubated in neutral red dye, before it is transferred to the recording chamber of the manual patch rig. The neutral red dye stains acidic components within the cell and enables the visualization of lysosomes which are suitable for extraction and patching.
- The coverslip is then scanned for a lysosome that is larger than the diameter of the pipette tip, and close to the edge of a cell so that it can easily be extracted and patched.
- A relatively sharp pipette is used to slice the cell membrane to create a rupture point through which the lysosome can be squeezed
- A fresh fire polished pipette is then used to patch the lysosome. It is filled with intracellular solution, placed on the pipette holder, put into the solution chamber, and used to approach the lysosome on the coverslip.
- When a gigaseal has been formed on the lysosome, a zap or voltage pulse is used to break in and achieve the whole-lysosome configuration.
See this in action in our video demonstrating the lysosomal patch-clamp technique to study ion channels on the lysosomal membrane.
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