NT5E KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 121 bp deletion in exon 2.
Images
Key facts
- Cell type
- A-375
- Species or organism
- Human
- Tissue
- Skin
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 121 bp deletion in exon 2
Alternative names
5' nucleotidase (CD73), 5' nucleotidase precursor, 5' nucleotidase, ecto, 5' nucleotidase, ecto (CD73), 5'-NT, 5'-nucleotidase, 5NTD_HUMAN, CD73, CD73 antigen, E5NT, Ecto-5'-nucleotidase, NT, NT5, NT5E, Purine 5 Prime Nucleotidase, eN, eNT
Recommended products
NT5E KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 121 bp deletion in exon 2.
Key facts
- Cell type
- A-375
- Species or organism
- Human
- Tissue
- Skin
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 121 bp deletion in exon 2
- Disease
- Melanoma
- Concentration
Properties
- Gene name
- NT5E
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing, Western blot
- Zygosity
- Homozygous
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 1 US: 1
- Adherent/suspension
- Adherent
- Gender
- Female
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.
- Culture medium
- DMEM (High Glucose) + 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
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
CD73 also known as ecto-5'-nucleotidase is an enzyme that functions at the cellular level. The CD73 protein hydrolyzes extracellular nucleotides into nucleosides by removing phosphate groups. It is a 70 kDa glycosylphosphatidylinositol-anchored molecule found on the surface of various cell types including endothelial cells lymphocytes and epithelial cells. Its presence is noted in a wide range of tissues across the human body highlighting its versatile role in cellular communication and metabolism.
- Biological function summary
CD73 participates in the regulation of purinergic signaling through adenosine production. It acts in cooperation with other cell surface enzymes such as CD39 forming a functional complex that handles ATP breakdown to adenosine. This enzymatic activity modulates immune responses tissue protection and inflammation control. In addition to immune-related functions CD73 supports the maintenance of vascular integrity and proper cellular adhesion which is important in different physiological and pathological contexts.
- Pathways
CD73 plays an important part in the adenosinergic pathway governing the conversion of AMP into adenosine. This activity impacts the signaling and function of the adenosine receptors on immune and non-immune cells. Another important pathway is the angiogenesis process where CD73 contributes by affecting endothelial cell migration and blood vessel development. Relationships with key proteins such as adenosine A2 receptors help facilitate these critical pathway activities.
- Associated diseases and disorders
CD73's involvement with immunosuppression and tumor progression becomes significant. Elevated levels of CD73 are frequently observed in some cancers where it aids in creating a favorable tumor microenvironment through increased adenosine production. This situation assists tumor cells in evading immune detection. Additionally CD73 is linked to autoimmune diseases like multiple sclerosis where it contributes to modulated lymphocyte activity. Connections with proteins such as TGF-beta further illustrate its role in disease-related pathways creating potential targets for therapeutic interventions through the use of anti-CD73 antibodies.
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3 product images
Western blot - Human NT5E knockout A375 cell line (ab281591)
False colour image of Western blot: Anti-CD73 antibody [4G6E3] staining at 1/500 dilution shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-CD73 antibody [4G6E3] ab202122 was shown to bind specifically to CD73. A band was observed at 65-70 kDa in wild-type A-375 cell lysates with no signal observed at this size in NT5E knockout cell line ab281591 (knockout cell lysate Human NT5E knockout A375 cell lysate ab282943). A cross-reactive band is observed at 65 kDa in all samples. To generate this image wild-type and NT5E knockout A-375 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 °. 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
All lanes: Western blot - Anti-CD73 antibody [4G6E3] (Anti-CD73 antibody [4G6E3] ab202122) at 1/500 dilution
Lane 1: Wild-type A-375 cell lysate at 20 µg
Lane 2: NT5E knockout A-375 cell lysate at 20 µg
Lane 2: Western blot - Human NT5E knockout A375 cell line (ab281591)
Lane 3: A431 cell lysate at 20 µg
Lane 4: HepG2 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 63 kDa
Observed band size: 65 kDa, 65-70 kDa
Sanger Sequencing - Human NT5E knockout A375 cell line (ab281591)
121 bp deletion in exon 2
Western blot - Human NT5E knockout A375 cell line (ab281591)
All lanes: Western blot - Anti-CD73 antibody [EPR6114] (Anti-CD73 antibody [EPR6114] ab133582) at 1/1000 dilution
Lane 1: Wild-type A-375 cell lysate at 20 µg
Lane 2: NT5E knockout A-375 cell lysate at 20 µg
Lane 3: A431 cell lysate at 20 µg
Lane 4: HepG2 cell lysate at 20 µg
SecondaryLanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 70 kDa
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