HLA-A KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided.
Images
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Sanger Sequencing, Western blot
Alternative names
Antigen Presenting Molecule, HLA class I histocompatibility antigen, A 1 alpha chain, Leukocyte antigen class I A, MHC class I antigen HLA A heavy chain, Major histocompatibility complex, class I, A
Recommended products
HLA-A KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided.
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Sanger Sequencing, Western blot
- Disease
- Carcinoma
- Concentration
Properties
- Gene name
- HLA-A
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Next Generation Sequencing, Sanger Sequencing, Western blot
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 1 US: 1
- Adherent/suspension
- Adherent
- Gender
- Male
Handling procedures
- Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
- Culture medium
- F-12K + 10% FBS
- Cryopreservation medium
- Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
Recommended control: Human wild-type A549 cell line (ab288558). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 10% FBS
Initial handling guidelines:
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x103-1x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.
Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 6x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
HLA-A also known as Human Leukocyte Antigen A is a protein that is part of the major histocompatibility complex (MHC) class I molecules. It has an approximate molecular mass of 44 kDa. HLA-A is expressed on the surface of nearly all nucleated cells and interacts with CD8+ T lymphocytes. Known for its role in immune recognition HLA-A presents endogenous peptide antigens to the T-cell receptor on cytotoxic T cells initiating immune responses to eliminate infected or malignant cells.
- Biological function summary
This target functions as a component of the MHC class I molecule complex. HLA-A binds peptides derived from proteasomal degradation of intracellular proteins and presents these peptides on the cell surface. The function plays an essential role in immune surveillance and distinction between self and non-self. This antigen presentation is important for immune responses against viral infections and tumor surveillance influencing the activation and proliferation of T lymphocytes.
- Pathways
HLA-A plays a significant role in the antigen processing and presentation pathway. It is closely related to the TAP (Transporter associated with Antigen Processing) proteins which transport antigenic peptides into the endoplasmic reticulum for loading onto MHC class I molecules. Additionally the target interacts with the CD8 molecule on cytotoxic T cells. This interaction is important within the immune response pathway contributing to the recognition and destruction of infected cells.
- Associated diseases and disorders
HLA-A has been associated with autoimmune diseases and transplant rejection. It plays a significant role in conditions like ankylosing spondylitis where specific HLA-A subtypes contribute to disease susceptibility. Additionally mismatches in HLA-A typing can lead to transplant rejection. In such scenarios anti-HLA antibodies can develop causing damage to transplant tissues. The complex interaction of HLA-A with related proteins such as other MHC class I molecules influences immune response outcomes in these diseases.
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3 product images
Next Generation Sequencing - Human HLA-A knockout A549 cell line (ab287473)
185 bp deletion after Gly123 of the WT protein
Western blot - Human HLA-A knockout A549 cell line (ab287473)
Anti-HLA-A antibody [EP1395Y] (Anti-HLA A antibody [EP1395Y] ab52922) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-HLA A antibody [EP1395Y] ab52922 was shown to bind specifically to HLA-A. A band was observed at 41 kDa in wild-type A549 cell lysates with no signal observed at this size in HLA-A knockout cell line. To generate this image, wild-type and HLA-A knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-HLA A antibody [EP1395Y] (Anti-HLA A antibody [EP1395Y] ab52922) at 1/10000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: Western blot - Human HLA-A knockout A549 cell line (ab287473)
Lane 2: HLA-A knockout A549 cell lysate at 20 µg
Lane 3: Wild-type A431 cell lysate at 20 µg
Lane 4: HLA-A knockout A431 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 41 kDa
Observed band size: 41 kDa
Sanger Sequencing - Human HLA-A knockout A549 cell line (ab287473)
185bp after Gly 124 in WT protein.
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