KC-3034
K562-HLA-A0101 Cell Line
Background of K562-HLA-A0101 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-3034 |
|---|---|
| Cell Line Name | K562-HLA-A0101 Cell Line |
| Host Cell Line | K562 |
| Description | Stable K562 clone expressing exogenous HLA-A0101 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% RPMI1640+20% FBS+10% DMSO |
| Propagation Medium | RPMI1640 + 10% FBS + 300 μg/mL Hygromycin B |
| Selection Marker | Hygromycin B |
| Morphology | Mostly single, round (some polymorph) cells in suspension |
| Subculture | Split saturated culture 1:4-1:8 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
K562 HLA-A0101 cell line was generated using a lentiviral vector expressing the HLA-A0101 sequence.
Characterization
Figure 1: Characterization of HLA-A0101 overexpression in K562 stable clone using FACS.
Figure2: Characterization of HLA-A0101 and its mutants overexpressing in K562 stable clone using PCR sequencing.
Cell Resuscitation
1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 300 μg/mL Hygromycin B)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL.
Cell Freezing
1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.
References
1.Huang X, Kushekhar K, Nolte I, Kooistra W, Visser L, Bouwman I, Kouprie N, Veenstra R, van Imhoff G, Olver B, Houlston RS, Poppema S, Diepstra A, Hepkema B, van den Berg A. HLA associations in classical Hodgkin lymphoma: EBV status matters. PLoS One. 2012;7(7):e39986. doi: 10.1371/journal.pone.0039986. Epub 2012 Jul 10. PMID: 22808081; PMCID: PMC3393726.
2.Henry JB, Hubbell CA, Davis MC, Fernandez-Vina MA, Yunis EJ, Shrimpton AE. A new HLA-A1 mutation: a novel, null variant allele. Am J Clin Pathol. 2004 Aug;122(2):185-92. doi: 10.1309/8DE1-2YU6-5BKH-HQW6. PMID: 15323134.
2.Henry JB, Hubbell CA, Davis MC, Fernandez-Vina MA, Yunis EJ, Shrimpton AE. A new HLA-A1 mutation: a novel, null variant allele. Am J Clin Pathol. 2004 Aug;122(2):185-92. doi: 10.1309/8DE1-2YU6-5BKH-HQW6. PMID: 15323134.