Neuronal Basal Medium (L-Glutamine-Free) 

MCE Neuronal Basal Medium (L-Glutamine-Free) is a culture medium specifically designed for the in vitro culture of primary or embryonic neuronal cells. When supplemented with Bi-27 (MCE Cat. No. HY-K3013/A) or N-2 (MCE Cat. No. HY-K3012/A) additives, it can be used for the cultivation of hippocampal neurons, cortical neurons, and neurons from other regions of the brain. It can also support the short-term or long-term survival and growth of homogenous neuronal populations in the absence of feeder cells.

This product is a serum-free medium, prepared with water-for-injection, containing essential and non-essential amino acids, vitamins, etc., but it does not contain L-Glutamine, L-Glutamic acid, and L-Aspartic acid (Formulation provided in the appendix).

4℃, 1 year

Culture containers and equipment: culture plates, culture flasks, and CO₂ incubators.

N-2 Supplement (HY-K3012/A) or Bi-27 Supplement (HY-K3013/A)

L-Glutamine (HY-K1046)

Poly D-Lysine solution (0.05 mg/mL)

1. This product is a serum-free medium. To prepare complete neuronal culture medium, it is recommended to choose one of the following formulations.

2. For the initial plating of primary neuronal cells, it is recommended to supplement the medium with 25 μM L-glutamic acid. Some cell lines may require 2% serum to promote cell attachment.

Note: Glutamate is required only for the initial plating of primary hippocampal cells. It is not necessary to add it during subsequent passaging or culture.

3. When culturing hippocampal neurons, it is recommended to add β-mercaptoethanol to extend the cell viability, with a final concentration of 25 μM.

1. Plate Pre-Treatment

1) Coat the 48-well culture plate or other culture vessels with pre-cooled Poly D-lysine (0.5 mg/mL). The recommended coating volume is 0.15 mL/cm². Incubate at room temperature for 1 h.

Note: Poly D-lysine enhances cell adhesion by altering the surface charge of the culture dish. It also improves the adsorption of serum proteins and extracellular matrix proteins, and increases the survival rate of subsequently cultured primary neurons.

2) Remove the coating solution and rinse the wells twice with sterile water.

Note: Coating solution may be cytotoxic, so thorough rinsing is necessary.

3) After rinsing, do not cover the plate. Leave it at room temperature to air dry completely. The dried plate can be used immediately or stored in a dry environment at 4°C for up to two weeks.

2. Cell Seeding

Add 60 - 150 µL of pre-warmed complete neuronal culture medium to each well and seed the cells at a density of 90 - 320 cells/mm². Incubate at 37°C with 5% CO₂ for 1 h.

Note: Complete neuronal cell culture medium can be preheated at 37°C in the incubator during inoculation.

3. Medium Replacement

Tilt the culture plate and gently aspirate the medium. Immediately add pre-warmed complete neuronal culture medium. The recommended volume is 0.4 mL per well. Continue incubation at 37°C with 5% CO₂.

4. Cell Culture Maintenance

Perform the first medium change 3 - 4 days after seeding. Thereafter, replace the medium every 3 days. It is recommended to use a half-medium change method-remove half of the old medium and replace with an equal volume of fresh medium.

Note: When culturing neuroblastoma cells, L-glutamine must be included in both the seeding and maintenance media.

Primary neurons are extremely fragile after thawing from cryopreservation. Do not centrifuge the cells.

1. Preparation of Experimental Materials

Since neurons can adhere to plastic and glass surfaces, to maximize cell recovery, it is recommended to rinse all materials (e.g., pipette tips, centrifuge tubes) with complete neuronal culture medium before starting the procedure.

2. Plate Pre-Treatment

Pre-coat the 48-well plate with pre-chilled poly D-lysine (0.5 mg/mL) as described above.

3. Medium Preparation

Pre-warm the complete neuronal culture medium in a 37°C incubator before use.

4. Cell Thawing

Place the cryovial in a 37°C water bath. As soon as the contents are thawed, immediately remove the vial from the bath. Total thawing time should be less than 1 min.

5. Cell Collection

1) Rinse the pipette tip with complete neuronal culture medium. Gently transfer the thawed cell suspension into a 15 mL centrifuge tube.

2) Rinse the cryovial with 1 mL of pre-warmed complete neuronal culture medium. Slowly add the rinse to the centrifuge tube dropwise at a rate of 2 drops/s, gently mixing after each drop.

3) Continue to add pre-warmed complete medium dropwise with gentle mixing after each drop until the final volume reaches 4 mL.

4) Perform cell counting.

6. Cell Seeding

Seed 1 × 10⁵ cells per well into the pre-treated 48-well plate. Add complete neuronal culture medium to bring the final volume to 0.5 mL per well. Incubate the plate at 37°C, 5% CO₂.

7. Cell Culture

Change the medium every 3 days. Use a half-medium change method: remove half of the medium and replace it with an equal volume of fresh medium (L-glutamic acid Free).

1. This product is a sterile solution, pay attention to aseptic operation to avoid contamination.

2. This product is for R&D use only, not for drug, household, or other uses.

3. For your safety and health, please wear a lab coat and disposable gloves to operate.