KC-3903

A375-HLA-A0201 Cell Line

33914

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Background of A375-HLA-A0201 Cell Line

HLA-A belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. They are expressed in nearly all cells. The heavy chain is approximately 45 kDa and its gene contains 8 exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode the alpha1 and alpha2 domains, which both bind the peptide, exon 4 encodes the alpha3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail. Polymorphisms within exon 2 and exon 3 are responsible for the peptide binding specificity of each class one molecule. Typing for these polymorphisms is routinely done for bone marrow and kidney transplantation. More than 6000 HLA-A alleles have been described. The HLA system plays an important role in the occurrence and outcome of infectious diseases, including those caused by the malaria parasite, the human immunodeficiency virus (HIV), and the severe acute respiratory syndrome coronavirus (SARS-CoV). The structural spike and the nucleocapsid proteins of the novel coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19), are reported to contain multiple Class I epitopes with predicted HLA restrictions. Individual HLA genetic variation may help explain different immune responses to a virus across a population.

Specifications

Catalog Number	KC-3903
Cell Line Name	A375-HLA-A0201 Cell Line
Clone Number	1#
Host Cell Line	A375
Description	Stable A375 cell line expressing exogenous HLA-A0201 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 + 100ug/ml HygromycinB
Selection Marker	Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:4 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

A375 HLA-A0201 cell line was generated using a lentiviral vector expressing the HLA-A0201 sequence.

Characterization

Figure 1: Characterization of HLA-A0201 overexpression in A375 HLA-A0201 stable clone using FACS.

Figure 2: Characterization of HLA-A0201 overexpressing in A375 HLA-A0201 stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 100ug/ml HygromycinB)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 1:2-1:4 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

Di D, Nunes JM, Jiang W, Sanchez-Mazas A. Like Wings of a Bird: Functional Divergence and Complementarity between HLA-A and HLA-B Molecules. Mol Biol Evol. 2021 Apr 13;38(4):1580-1594.
Yu H, Ye C, Li J, Pan C, Lin W, Chen H, Zhou Z, Ye Y. An altered HLA-A0201-restricted MUC1 epitope that could induce more efficient anti-tumor effects against gastric cancer. Exp Cell Res. 2020 May 1;390(1):111953.
Zhu K, Lizee G, Cano P, Fernando-Vina M, Ji B, Abbruzzese JL, Hwu P, Radvanyi L, Chang DZ. HLA-A0201 positive pancreatic cell lines: new findings and discrepancies. Cancer Immunol Immunother. 2007 May;56(5):719-24.