KC-6183

HPB-ALL-CD8a-KO Cell Line

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Home » HPB-ALL-CD8a-KO Cell Line

Background of HPB-ALL-CD8a-KO Cell Line

CD8A (CD8 Subunit Alpha) is a Protein Coding gene. This gene encodes the alpha chain of CD8, a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The encoded protein is a member of the immunoglobulin superfamily and functions as a coreceptor alongside the T-cell receptor (TCR) to recognize antigens presented by antigen-presenting cells in the context of class I MHC molecules. The CD8 coreceptor can exist as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Upon TCR recognition of MHC class I:peptide complexes, CD8A interacts with the MHC class I protein and recruits the Src kinase LCK to the vicinity of the TCR-CD3 complex, initiating downstream intracellular signaling pathways that ultimately lead to lymphokine production, motility, adhesion, and activation of cytotoxic T-lymphocytes (CTLs). This mechanism enables CTLs to recognize and eliminate infected cells and tumor cells. In NK-cells, CD8A homodimers at the cell surface provide a survival mechanism allowing conjugation and lysis of multiple target cells. Mutations in this gene are associated with immunodeficiency 116 (IMD116). Multiple transcript variants encoding different isoforms have been found for this gene, with the major protein isoforms differing by the presence or absence of a transmembrane domain.

Specifications

Catalog NumberKC-6183
Cell Line NameHPB-ALL-CD8a-KO Cell Line
Clone Number2C2
Host Cell LineHPB-ALL
DescriptionStable HPB-ALL-CD8a-KO clone with CD8a gene knockout, No.2C2
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640+20% FBS+10% DMSO
Propagation Medium90% RPMI1640+10% FBS
Selection MarkerNA
Morphologylymphoblast
SubcultureSplit saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 50 hours
Mycoplasma StatusNegative

Cell Line Generation

HPB-ALL-CD8a-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HPB-ALL-CD8a-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of HPB-ALL-CD8a-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of HPB-ALL-CD8a-KO cell line stable clone using FACS.

Cell Resuscitation

  1. Prewarm culture medium (90% RPMI1640+10% FBS) 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 1:3-1:6 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.

References

  1. Clark EA, Ledbetter JA. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50. Proc Natl Acad Sci U S A. 1986;83(12):4494-4498. doi:10.1073/pnas.83.12.4494. PMID: 2424037; PMCID: PMC323754.
  2. Gao GF, Rao Z, Bell JI. Molecular coordination of alphabeta T cell receptors and coreceptors CD8 and CD4 in their recognition of peptide-MHC ligands. Trends Immunol. 2002;23(8):408-413. doi:10.1016/S1471-4906(02)02282-2. PMID: 12133802.

Use License Agreement

Research Use Only.
Not for use in diagnostic procedures or therapeutic applications.
Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.
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