HT-22 Cell Line

Brief Introduction
Product List

General Information
Organism	Mus musculus, mouse
Cell Line Description	HT-22 is an immortalized mouse hippocampal neuronal cell line established by the laboratory of David Schubert at the Salk Institute. This cell line is derived from a stable subclone of the parental HT-4 lineage; the HT-4 precursor cells were originally generated by immortalizing mouse neuronal tissue using the temperature-sensitive Simian Virus 40 (SV40) large T antigen. HT-22 cells grow as an adherent monolayer and exhibit a distinct, differentiated neuronal phenotype. In the fields of neurobiology and pharmacology, HT-22 is recognized as the "gold standard" and preferred in vitro model for studying glutamate-induced oxidative toxicity (oxytosis) and ferroptosis. Because these cells lack functional ionotropic N-methyl-D-aspartate (NMDA) receptors, the mechanism of glutamate-induced cell death relies entirely on the competitive inhibition of the cystine/glutamate antiporter, leading to intracellular glutathione depletion and triggering severe lipid peroxidation. This makes HT-22 an indispensable tool for elucidating pathways associated with neurodegenerative diseases (such as Alzheimer's and Parkinson's diseases) and for screening protective compounds.
Tissue	Brain; derived from Hippocampus
Cell Type	Neuronal cell
Disease	Normal / Non-malignant tissue origin (Immortalized engineered line)
Morphology	Neuronal / Epithelial-like with fine cytoplasmic extensions
Growth Mode	Adherent Monolayer
Biosafety Level	1 (Biosafety classification aligns with global standard laboratory parameters for non-infectious animal materials)
Applications	1. Investigating "gold-standard" in vitro models of glutamate-induced oxidative stress, glutathione depletion, and mitochondrial reactive oxygen species (ROS) generation; 2. Evaluating standard marker panels for lipid peroxidation-dependent ferroptosis signaling; 3. Screening neuroprotective drug candidates for stroke, traumatic brain injury, and age-related cognitive decline; 4. Elucidating the molecular cascades of non-receptor-mediated cell death (oxytosis); 5. Assessing downstream epigenetic alterations, chromatin remodeling, and transcriptional responses following severe neurotoxic injury.
Shipped In	Dry ice
Storage Temperature	−196°C
Characteristics
Tumorigenic	No, natively non-tumorigenic under standard physiological baselines.
Transformant	Temperature-sensitive Simian virus 40 (SV40) large T-antigen.
Unique Mechanism	HT-22 cells lack functional NMDA receptors. When exposed to millimolar concentrations of extracellular glutamate, these cells do not undergo conventional receptor-mediated excitotoxicity; instead, the exposure triggers a competitive blockade of cystine uptake. This process leads to the rapid depletion of intracellular glutathione pools, subsequently inducing mitochondrial calcium overload, the accumulation of reactive oxygen species (ROS), and programmed ferroptosis.
Growth Kinetics	Exceptionally rapid adherent proliferation; typical population doubling time is approximately 15 hours.
Mycoplasma Test	Negative
Culture Conditions and Handling
Subculturing	1. Remove and discard all spent complete culture medium from the culture dish. 2. Briefly rinse the cell monolayer with sterile, Ca2+/Mg2+-free PBS buffer to remove residual serum. 3. Add 1.0 to 2.0 mL of pre-warmed Accutase (preferred) or 0.25% Trypsin-0.53 mM EDTA solution, ensuring the cell layer is completely covered. 4. Incubate at room temperature or 37°C for 2 to 5 minutes. Monitor closely under an inverted microscope until the cells become rounded, detach, and slide easily off the surface. 5. Immediately add 6.0 to 8.0 mL of pre-warmed complete culture medium to the dish to neutralize enzymatic activity. 6. Gently pipette the suspension up and down to achieve a uniform single-cell suspension. Centrifuge at approximately 300 × g for 5 minutes, carefully aspirate the supernatant, gently resuspend the cells in complete culture medium, and distribute them into new vessels.
Thawing Protocol Note	HT-22 cells are sensitive to pH levels and mechanical agitation during the initial recovery phase following thawing. Before adding the thawed cell suspension, the culture vessel containing fresh complete growth medium should be placed in a 5% CO2 incubator for at least 15 minutes to allow the medium to reach and equilibrate within the normal physiological pH range (7.0 to 7.6).
Medium Renewal	2 to 3 times per week
Subcultivation Ratio	Split optimal, sub-confluent cultures (70-80% density) at a standard ratio of 1:3 to 1:6. Avoid letting the cells reach 100% confluence to preserve their phenotype.
Culture Conditions	Atmosphere: Air, 95%; CO2, 5%; Temperature: 37°C
Cryopreservation	90% Complete culture growth medium + 10% DMSO

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* For research use only. Not intended for any clinical use.

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