Protocol Guide: Spiny Striatal GABAergic Neuron Monoculture

Spiny Striatal GABAergic Neurons

Spiny striatal GABAergic neurons are inhibitory neurons of a striatal lineage that are found in the striatum. These neurons release GABA as their primary inhibitory neurotransmitter and are important for proper neurologic function. Through our partnership with BrainXell, we offer fully differentiated spiny striatal GABAergic neurons that express GABA/DARPP32/MAP2 at DIV 7 with spontaneous firing by DIV 21. Spiny GABAergic neurons are often used in vitro to study Huntington’s disease, Parkinson’s disease, and schizophrenia.

We describe the seeding and culturing protocol for spiny striatal GABAergic neurons in monoculture below.

Section Overview

• Spiny GABAergic Neuron Culture Materials
• Spiny GABAergic Neuron Culture Protocol

Spiny GABAergic Neuron Culture Materials

Neuron cryovials must be stored immediately a liquid or vape nitrogen storage system. Store the BrainFast supplements at -80°C for up to 18 months or -20°C for up to 6 months.

• BrainXell Human Medium Spiny GABAergic Neurons (BX-0700-30-1M)
• BrainFast GABA Seeding Supplement (BX-2400-100uL)
• BrainFast D4/Day 4 Supplement (BX-2040-100uL)
• BrainFast SK/Supplement K (BX-2020-100uL)
• DMEM/F12 Medium, +L-glutamine, +HEPES (D0697)
• Neurobasal Medium (Thermo Fisher Scientific #21103049)
• B-27 Supplement (Thermo Fisher Scientific #17504044)
• N-2 Supplement (Thermo Fisher Scientific #17502048)
• GlutaMAX Supplement (Thermo Fisher Scientific #35050061)
• Cultrex (R&D Systems #3432-001-01)
• Ascorbic Acid (A8960)
• PDL-coated 96-Well Plate (M0812, LPDL001)

Spiny GABAergic Neuron Culture Protocol

Day 0: Seeding Preparation

You will need to seed 4.4 - 5.5 million live cells for a 96-well plate, but can use additional cells depending on experimental needs. For further viability and seeding information, see the Certificate of Analysis.

1. Using the components listed below make the Basal Medium in the cell culture hood or biological safety cabinet. Do not place the medium in a 37°C water bath but allow medium to come to room temperature for 15 minutes prior to use. Store at 4°C for up to 3 weeks.
   1. Growth factors are optional – recommended final concentrations are: 10µg/ml GDNF, 2µg/ml TGF-β1, and 10µg/ml BDNF.

*For 200mM Ascorbic Acid Solution preparation, click here.

2. Prepare a Cultrex solution by adding 495µl of cold DMEM/F12 medium to a 55µl aliquot of frozen Cultrex from the -80°C freezer. Mix the solution to dissolve and store at 4°C.
3. Prepare cell counting solutions by adding 25µl of Trypan Blue solution to a microcentrifuge tube 3ml of Basal Medium to a separate 50ml conical tube.

Day 0: Seeding the Spiny GABAergic Neurons

1. Place a cryovial of spiny GABAergic neurons in a 37°C water bath to thaw. Avoid submerging the cap to minimize contamination.
2. Remove the vial from the water bath as soon as the last ice melts (~75-90 seconds) and disinfect the vial with 70% ethanol. Transfer the vial to the cell culture hood.
3. Transfer 500µl, using a P1000 pipette at a rate of 2-3 drops per second, of the Basal Medium from the 50ml conical tube to the spiny GABAergic neuron vial. This transfer process should take about 10 seconds.
4. Transfer the contents of the cryovial (~1ml) to the 50ml Basal Medium conical tube.
5. Centrifuge the cells for 5 minutes at 465xg.
6. Aspirate the supernatant carefully. Resuspend the cell pellet in 950µl of fresh Basal Medium by gently pipette up and down using a P1000 pipette.
7. Resuspend the cells to 1.0 x 10⁶ live cells/ml final concentration based on the value of viable cells/vial (from the CoA). Add Basal Medium slowly to the existing 1ml in the conical tube to get to the desired concentration.
   1. For example: dilute 5.3 million viable cells/vial to 5.3 total volume.
8. Count the cells using the Trypan Blue solution from Step 3. Evenly suspend cells in Basal Medium by pipetting up and down 3-5 times. Transfer 25µl of the cell suspension to the 25µl Trypan Blue solution microcentrifuge tube. Mix by pipetting. Using a hemocytometer, count the number of viable and dead cells and determine the live cell concentration (live cells/ml).
9. Calculate the volume needed to create Medium Spiny Neuron (MSN) Seeding Suspension. The typical seeding density for MSNs is 40,000-50,000 viable cells/100µl/well for a 96-well plate i.e. ~125,000-156,250 viable cells/cm², but the recommended seeding density may vary so refer to the CoA for lot-specific seeding information. Dilute the cells to the desired seeding concentration based on Trypan Blue cell count taken above.

Example of dilution calculation:

10. In a new, sterile 50ml conical tube mix the previously calculated volumes of the Basal Medium and the Spiny GABAergic neurons to obtain 11ml of the MSN seeding suspension.
11. Make the MSN Seeding Medium for the Spiny GABAergic neurons:

12. Completely mix and transfer 100µl/well (40,000 cels/well) of the MSN Seeding Medium to a PDL-coated 96-well plate. Do NOT agitate the plate during the seeding process - plate movement can lead to uneven cell attachment.
13. Allow plate to rest after seeding for 10 minutes for cells to settle to the bottom of the well before moving. Transfer MSN plate to a humidified incubator set to 37°C with 5% CO₂.

This thawing and plating protocol should not exceed 1 hour. The post-thaw viability and health of the spiny GABAergic neurons can be negatively impacted if the process takes too long.

Day 1: Spiny GABAergic Neuron Medium Replacement

1. On Day 1, prepare Day 1 Medium using the below components:

2. Remove the 100µl currently in each well and replace with 100µl Day 1 Medium per well. Complete one column or row at a time to prevent the wells from drying out.

Day 4: Spiny GABAergic Neuron Medium Replacement

1. On Day 4, prepare Day 4 Medium using the below components:

2. Add 100µl of Day 4 Medium to the entire plate; there should be a final volume of 200µl Medium per well.

Day 7 and Onward: Spiny GABAergic Neuron Medium Changes

1. Change half to media weekly using the Basal Medium, i.e. on Day 7, 14, 21, etc., by removing 100µl/well and adding 100µl/well of Basal Medium to the plate.

NOTE: Adding BrainFast SK at low concentrations (0.1-0.5X) in the medium may be helpful for long-duration cultures.

Spiny GABAergic neurons mature rapidly and can be maintained in culture for at least 3 weeks post-seeding under the above conditions.

These products are available in only the US and UK.