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AB281591

Human NT5E knockout A375 cell line

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NT5E KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 121 bp deletion in exon 2. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
3 Images
Western blot - Human NT5E knockout A375 cell line (AB281591)
  • WB

Lab

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] (ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot 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 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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-CD73 antibody [4G6E3] (<a href='/en-us/products/primary-antibodies/cd73-antibody-4g6e3-ab202122'>ab202122</a>) 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

Predicted band size: 63 kDa

Observed band size: 65 kDa,65-70 kDa

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Western blot - Human NT5E knockout A375 cell line (AB281591)
  • WB

Lab

Western blot - Human NT5E knockout A375 cell line (AB281591)

All lanes:

Western blot - Anti-CD73 antibody [EPR6114] (<a href='/en-us/products/primary-antibodies/cd73-antibody-epr6114-ab133582'>ab133582</a>) 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

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Observed band size: 70 kDa

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Sanger Sequencing - Human NT5E knockout A375 cell line (AB281591)
  • Sanger seq

Lab

Sanger Sequencing - Human NT5E knockout A375 cell line (AB281591)

121 bp deletion in exon 2

Key facts

Cell type

A-375

Species or organism

Human

Tissue

Skin

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 121 bp deletion in exon 2

Disease

Melanoma

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Complementary Products

Primary Antibodies

AB288154

Anti-CD73 antibody [EPR25191-104]

20ul selling size|RabMAb|Recombinant

Flow Cytometry - Anti-CD73 antibody [EPR25191-104] (AB288154)

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Primary Antibodies

AB92574

Anti-CD90 / Thy1 antibody [EPR3132]

BOND RX™ Validated|20ul selling size|RabMAb|Recombinant|KO Validated

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD90 / Thy1 antibody [EPR3132] (AB92574)

  • 5
  • 6
Cell Lines & Lysates

AB262490

Human THY1 (CD90) knockout U-2 OS cell line

Advanced Validation

Western blot - Human THY1 (CD90) knockout U-2 OS cell line (AB262490)

  • 0
  • 0
Primary Antibodies

AB169545

Anti-CD105 antibody [EPR10145-12]

RabMAb|Recombinant|KO Validated

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD105 antibody [EPR10145-12] (AB169545)

  • 4
  • 3
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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "Sanger seq": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" }, "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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Product details

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.

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What's included?

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Properties and storage information

Gene name
NT5E
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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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.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

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.

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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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 1 US: 1

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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Target data

See full target information NT5E
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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.
For full details, please see our Terms & Conditions

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

For licensing inquiries, please contact partnerships@abcam.com