This article describes a method for isolating cholinergic neurons from transgenic Caenorhabditis elegans using fluorescence-activated cell sorting (FACS). The transgenic worms express a fluorescent protein that allows for the identification and separation of these neurons from other cell types.
Take a suspension of cells obtained from transgenic Caenorhabditis elegans worms in a culture medium.
The suspension contains cells from various tissues, including neurons from the nervous system.
The transgenic worms were engineered to express a fluorescent protein conjugated to a vesicular transmembrane transporter. This transporter, present on synaptic vesicles carrying the neurotransmitter acetylcholine, is found in cholinergic neurons throughout the body.
Centrifuge the cell suspension and discard the supernatant. Resuspend the cells in a fresh culture medium for fluorescence-activated cell sorting, or FACS.
Analyze the cell suspension using FACS. The fluorescently labeled cholinergic neurons emit fluorescence upon exposure to light of an appropriate wavelength, separating them from other unlabeled cells.
Transfer the isolated neurons into a multi-well plate. Incubate the cells in a humidified chamber to maintain the moisture level.
Centrifuge cells from the isolated cell suspension at 10,000 times g and at 4 degrees Celsius for 5 minutes. Discard the supernatant and resuspend the pelleted cells in L-15-supplemented medium to a cell density of no more than 6 million cells per milliliter to avoid overloading the flow cytometer. Then, sort cells by GFP-positive expression using a flow cytometer capable of sorting samples.
GFP-negative cells can be used as a control for non-cell-type-specific analysis. After this, plate the isolated cells into the wells of a sterile six-well plate. Place the plate into a plastic container. Add a damp wipe or soft tissue paper to add moisture to the cells and incubate at 20 degrees Celsius.
繁體中文
