Culturing Fluorescent Excitatory Neurons from a Transgenic Mouse Pup Brain

Samenvatting

Source: Turko, P., et al. Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting. J. Vis. Exp. (2019)

The video illustrates a technique for culturing fluorescent GABAergic and glutamatergic neurons from the cortex and hippocampal tissues of transgenic mouse pups. The process includes obtaining purified single-cell suspensions of neurons, sorting GABAergic neurons based on their fluorescence protein expression, and seeding them onto poly-L-lysine-coated plates to facilitate attachment and cell proliferation.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Coating Glass Coverslips with Poly-L-lysine

1. Prepare glass coverslips by defrosting a 5 mL aliquot of 200 µg/mL poly-L-lysine (PLL) solution. Dilute this stock solution to 20 µg/mL by adding 45 mL of pure injection grade water. Filter-sterilize the solution into a new 50 mL conical tube. Label this tube as "PLL Sterile".
   NOTE: Use water stored at room temperature to speed up the defrosting process.
2. Add 100 sterile, round, 12 mm glass coverslips to the ste....

Resultaten

Figure 1: Purification of glutamatergic and GABAergic neurons. (A) Images showing fluorescent signal from the transgene expression of TdTomato in NexCre;Ai9 mice (top) and Venus in VGAT Venus mice (bottom). Scale bars = 5 mm. (

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Neural Basal A media (NBA)	ThermoFisher Scientific	10888022	Cell Culture Buffer
B27	ThermoFisher Scientific	17504001	Culture supplement
Glutamax	ThermoFisher Scientific	35050-038	Culture supplement
Penicillin Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15140-122	Antibiotic
Poly-L-Lysine	SIGMA	P1399	Coverslip coating
Papain	SIGMA	P4762-1G	Enzyme
Bovine Serum Albumin	SIGMA	A3294-100G	Serum
Hibernate A low fluorescence media	Brain Bits Ltd	HALF	Cell Transport media
Fine Tip Pasteur Pipette	Neo Labs	-	Used for trituration of cells
24-well plates	BD	353047	Culture plate
50 mL Falcon tubes	BD	352070	-
15 mL Falcon tubes	BD	352096	-
Glass coverslips: 12 mm round	Roth	P231.1	-
35 mm Petri dish	Corning	353001	-
100 mm Petri dish	Corning	353003	-
30 µm CellTrics Cell Sieve	sysmex	04-004-2326	To remove cell clumps before cell sorting
Round bottom polystyrene tubes	BD	352054	Transport tube for sorted cells
Round bottom polypropylyne tubes	BD	352063	Collection tube for sorted cells
Extra fine Bonn Scissors	Fine Scientific Tools	14084-08	To remove overlying skin and bone of mice
Extra narrow Scissors	Fine Scientific Tools	14088-10	To remove overlying skin and bone of rats
Forceps	Fine Scientific Tools	11242-40	To hold the head in place
Spatula (130 mm long / 5 mm tip width)	Fine Scientific Tools	3006.1	To remove the brain to filter paper
Scalpel Blades	Swan-Morton	#0308	To mechanically dissociate neural tissue
Haemocytometer (Neubauer Imroved)	Optik Labor	-	To cell count dissociated cells