3A9 Cell Line

Brief Introduction
Product List

General Information
Organism	Mus musculus, mouse
Cell Line Description	3A9 is a murine T-cell hybridoma established in 1985 by Paul M. Allen, Emil R. Unanue, and their colleagues at Washington University School of Medicine. The cell line was generated via polyethylene glycol (PEG)-mediated somatic cell fusion, utilizing antigen-primed helper T lymphocytes from A/J mice immunized with hen egg-white lysozyme (HEL) and the TCR-deficient BW5147 thymoma fusion partner. 3A9 cells grow in suspension, appearing as round single cells or loose multicellular clusters. In the fields of immunology, antigen presentation, and T-cell signaling, 3A9 is a globally recognized, classic in vitro model for studying MHC class II-restricted CD4+ T-cell recognition. The cell line constitutively expresses a functional clonotypic T-cell receptor (TCR) that specifically recognizes the dominant HEL peptide epitope (HEL 46-61; sequence: NTDGSTDYGILQINSR) presented by the murine MHC class II molecule I-A^k. Upon antigen engagement, 3A9 cells secrete high levels of interleukin-2 (IL-2), making them a valuable tool for assessing antigen presentation efficiency, TCR-MHC interaction kinetics, and dendritic cell antigen-processing mechanisms.
Tissue	Blood / Lymphoid; T-cell Hybridoma (Fusion of helper T-lymphocytes and BW5147 thymoma)
Cell Type	CD4+ Helper T-Lymphocyte Hybridoma
Strain	A/J X AKR/J (Fusion between A/J T-cells and AKR-derived BW5147)
Disease	Normal / Antigen-specific Immune Cell (Hybridoma)
Morphology	Lymphoblast-like / Round cells in suspension
Product Format	Frozen
Growth Mode	Suspension
Biosafety Level	1 (Biosafety classification matches standard laboratory safety parameters for non-infectious rodent hybridoma lines)
Applications	1. An in vitro "gold standard" reporter system for determining the antigen presentation efficiency and processing kinetics of hen egg lysozyme (HEL 46-61); 2. Elucidating the molecular dynamics mechanisms underlying peptide loading, binding affinity, and TCR contact residues for MHC class II molecules (I-A^k); 3. Serving as an IL-2 reporter system (via ELISA or HT-2 bioassay) to quantitatively assess the activation capacity of dendritic cells, macrophages, or B cells; 4. Investigating T-cell receptor (TCR) activation, CD3 complex signal transduction, and immunological synapse formation; 5. Evaluating immunomodulatory compounds, MHC blockers, and adjuvant strategies targeting antigen-specific immune responses.
Shipped In	Dry ice
Storage Temperature	−196°C
Characteristics
Tumorigenic	No, typical T-cell hybridoma displaying infinite in vitro proliferative lifespan without tumorigenic engraftment properties in wild-type strains.
Transformant	None (Immortalized via somatic cell hybridization with the BW5147 thymoma line).
Antigen Specificity	Highly specific for the hen egg-white lysozyme (HEL) peptide sequence 46–61 (NTDGSTDYGILQINSR).
Phenotypic Markers	CD4-positive, CD3-positive, CD8-negative. Expresses functional surface TCR αβ chains specific for HEL/I-A^k.
Functional Readout	Antigen engagement of the surface TCR induces robust, quantifiable secretion of Interleukin-2 (IL-2) into the culture supernatant within 12 to 24 hours.
Mycoplasma Test	Negative
Culture Conditions and Handling
Subculturing	1. Since these cells grow entirely in suspension, no enzymatic treatment (such as trypsin digestion) is required. 2. Gently swirl or rotate the culture vessel to create a uniform cell suspension. 3. Transfer the cell-containing medium to a sterile conical centrifuge tube. 4. Centrifuge at approximately 125 × g (or 1000 rpm) for 5 minutes. 5. Carefully aspirate or decant the spent supernatant, taking care not to disturb the cell pellet at the bottom. 6. Gently resuspend the cell pellet using pre-warmed, fresh complete growth medium. 7. Critical density range: The initial seeding density should be at least 1.0 × 10⁵ to 2.0 × 10⁵ cells/mL. Maintain the cell density between 2.0 × 10⁵ and 1.0 × 10⁶ cells/mL during the active growth phase. Avoid exceeding a cell density of 1.5 × 10⁶ cells/mL to prevent nutrient depletion and sudden cell death.
Thawing Protocol Note	3A9 cells are relatively sensitive to pH fluctuations during the initial recovery phase after thawing. Before adding the thawed cell suspension, the culture flask containing fresh complete 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	Every 2 to 3 days
Subcultivation Ratio	Split saturated suspension cultures at a standard ratio of 1:3 to 1:10.
Culture Conditions	Atmosphere: Air, 95%; CO2, 5%; Temperature: 37°C
Cryopreservation	90% Complete culture growth medium + 10% DMSO (or 50% RPMI 1640 + 40% FBS + 10% DMSO)

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

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