Home › Neuroscience › Demonstration of Endo-Lysosomal Patch Clamp Technique
Steps
  1. 1 Seed cells on poly-D-lysine coverslips 00:14
  2. 2 Treat cells overnight with vacuolin-1 00:34
  3. 3 Stain lysosomes with neutral red dye 00:46
  4. 4 Locate and select target lysosomes 01:06
  5. 5 Slice membrane and extract lysosome 01:32
  6. 6 Form giga seal on lysosomal membrane 02:18
  7. 7 Achieve whole-lysosome configuration 02:52
Neuroscience Metrion Biosciences

Demonstration of Endo-Lysosomal Patch Clamp Technique

Protocol
Difficulty
intermediate

Steps

1
Seed cells on poly-D-lysine coverslips

Cells are seeded at high density onto poly-D-lysine coated coverslips to ensure proper adhesion and formation of a network that facilitates lysosome extraction.

▶ 00:14
2
Treat cells overnight with vacuolin-1

Cells are incubated overnight with the chemical vacuolin-1 to enlarge lysosomes to a size suitable for patch clamping.

▶ 00:34
3
Stain lysosomes with neutral red dye

The coverslip is incubated in neutral red dye to stain lysosomal compartments, enabling visualization of suitable lysosomes for extraction and patching.

▶ 00:46
4
Locate and select target lysosomes

The coverslip is scanned to identify suitable lysosomes that are larger than the pipette tip diameter and located near the cell edge for easy extraction.

▶ 01:06
5
Slice membrane and extract lysosome

A sharp pipette is used to slice the cell membrane creating a rupture point, then the lysosome is gently squeezed out through the opening.

▶ 01:32
6
Form giga seal on lysosomal membrane

A fire-polished pipette filled with intracellular solution is positioned against the extracted lysosome to form a high-resistance giga seal.

▶ 02:18
7
Achieve whole-lysosome configuration

A zap or voltage pulse is applied to rupture the lysosomal membrane and establish whole-lysosome recording configuration, confirmed by red dye leaching into the pipette.

▶ 02:52
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