KC-3127

293T-GRIN1-GRIN2B Cell Line

34154

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Background of 293T-GRIN1-GRIN2B Cell Line

NMDA receptors are the main excitatory amino acid receptors in the central nervous system, which are distributed in the whole brain, with the highest density in the hippocampus, cerebral cortex, striatum and amygdala. As an ionic receptor of excitatory amino acid glutamate in the central nervous system, the impairment of learning and memory seen in a variety of different pathologies, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease, Parkinson’s disease (PD), schizophrenia and major depressive disorder (MDD) are associated with NMDAR malfunction.Glutamate receptor ionotropic, NMDA 1 is a protein in humans that is encoded by GRIN1 gene. Component of NMDA receptor complexes that function as heterotetrameric, ligand-gated ion channels with high calcium permeability and voltage-dependent sensitivity to magnesium. Channel activation requires binding of the neurotransmitter glutamate to the epsilon subunit, glycine binding to the zeta subunit, plus membrane depolarization to eliminate channel inhibition by Mg2+.The GRIN2B gene encodes GluN2B. GluN2B is a subunit of the NMDA receptor (NMDAR). NMDARs are ionotropic glutamate receptors that play a crucial role in glutamatergic synaptic transmission, synaptic plasticity, and development. NMDARs have intrinsic ion channels that are permeable to sodium, potassium, and calcium.

Specifications

Catalog Number	KC-3127
Cell Line Name	293T-GRIN1-GRIN2B Cell Line
Clone Number	1-4#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous GRIN1 and GRIN2B gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +1μg/mL Puromycin+150μg/mL Hygromycin
Selection Marker	Puromycin \| Hygromycin B
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-GRIN1-GRIN2B cell line was generated using lentivirus expressing GRIN1 and GRIN2B sequence.

Characterization

Figure 1. Characterization of GRIN1 over-expression in the 293T-GRIN1-GRIN2B stable clone using qPCR.

Figure 2. Characterization of GRIN2B over-expression in the 293T-GRIN1-GRIN2B stable clone using qPCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 1μg/mL Puromycin+150μg/mL Hygromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Adell A. Brain NMDA Receptors in Schizophrenia and Depression. Biomolecules. 2020 Jun 23;10(6):947. doi: 10.3390/biom10060947. PMID: 32585886; PMCID: PMC7355879.
Jimenez EC. Peptide antagonists of NMDA receptors: Structure-activity relationships for potential therapeutics. Peptides. 2022 Jul;153:170796. doi: 10.1016/j.peptides.2022.170796. Epub 2022 Mar 30. PMID: 35367253.
Chen H, Dong Y, Wu Y, Yi F. Targeting NMDA receptor signaling for therapeutic intervention in brain disorders. Rev Neurosci. 2023 Jan 2;34(6):635-647. doi: 10.1515/revneuro-2022-0096. PMID: 36586105.