Culturing Fluorescent Excitatory Neurons from a Transgenic Mouse Pup Brain

First, dissect away the cerebellum and separate the two hemispheres. Then, carefully separate the hippocampus and cortex from each hemisphere. Transfer the dissected tissue to a 35-millimeter Petri dish containing chilled cell culture buffer.

Then, transfer the dissected hippocampus and cortex to the lid of another 35-millimeter Petri dish. Using the flat edge of a scalpel blade, carefully chop the tissue in a crisscross motion until only small pieces remain.

Next, transfer the chopped tissue with a small amount of papain solution from the Petri dish lid to the sterile papain tube. Incubate the tissue at 37 degrees Celsius for 25 minutes. After papain incubation, transfer the sterile papain tube and sterile BSA tubes to the flow cabinet. Then, use a 1-milliliter Pasteur pipette to transfer only the cortico-hippocampal tissue from the papain tube into the BSA tube 1.

In order to break up any large clumps of tissue, triturate the tissue several times using a 1-milliliter Pasteur pipette. Following this, triturate the tissue seven times using a fine-tip Pasteur pipette. After 30 seconds, transfer 1 milliliter of the lower solution and tissue from BSA tube 1 to BSA tube 2.

Triturate the tissue in BSA tube 2 several times using a fine-tip Pasteur pipette. After trituration, transfer 1 milliliter of the lower tissue in solution from BSA tube 1 to BSA tube 3. Triturate the tissue in BSA tube 3 several times.

After trituration, transfer all solution and tissue from tubes 2 and 3 into BSA tube 1. Triturate 2 to 3 more times and centrifuge at 3,000 times g for 3 minutes. Following centrifugation, carefully remove the supernatant from the pelleted tissue and resuspend the cells using a P1000 pipette in 2 milliliters of complete Hibernate A low-fluorescence medium. Then, triturate the tissue 20 times to ensure complete resuspension of the tissue solution.

Subsequently, filter the cell suspension through a 30-micrometer cell sieve into a polystyrene sample tube. Prepare cell-sorting collection tubes by pipetting 300 microliters of complete Hibernate A media to the required number of polypropylene tubes.

For each fluorescent cell type to be sorted, choose the appropriate excitation and emission filters. Excite venous protein using 488-nanometer excitation wavelength and detect the emitted light through 530 over 40 emission filter set.

Excite TdTomato protein using 531-nanometer excitation wavelength and detect the emitted light through 575 over 30 emission filter set. For high purity, sort brightly labeled fluorescent cells. Following cell sorting, transfer the collected cells to 2-milliliter round-bottom centrifuge tubes. Then, centrifuge the cells at 3,000 times g for 3 minutes to form a cell pellet.

Resuspend the cell pellet in the required amount of pre-warmed complete NBA medium to achieve a cell density of 1,000 cells per microliter. To confirm the presence of dissociated cells, check the cell solution under a microscope using a 4x or a 10x objective lens. Before plating the cells, vortex at a medium speed for 2 to 3 seconds to ensure an even cell suspension.

Following vortexing, quickly pipette 10 microliters of the cell suspension to the center of each coverslip. After one hour, feed the cells with 500 microliters of pre-warmed complete NBA medium and return to the incubator at 37 degrees Celsius.