CD47 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 2 and 5 bp deletion in exon 2.
Images
Key facts
- Cell type
HEK-293T
- Species or organism
Human
- Tissue
Kidney
- Form
Liquid
- Knockout validation
Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 2 and 5 bp deletion in exon 2
Alternative names
Antigen identified by monoclonal antibody 1D8, Antigenic surface determinant protein OA3, CD47 antigen, CD47 antigen (Rh-related antigen, integrin-associated signal transducer), CD47 glycoprotein, CD47 molecule, CD47_HUMAN, IAP, Integrin associated signal transducer, Integrin-associated protein, Leukocyte surface antigen CD47, MER 6, OA 3, OTTHUMP00000041152, OTTHUMP00000041153, Protein MER6, Rh related antigen, Surface antigen identified by monoclonal antibody 1D8
Recommended products
CD47 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 2 and 5 bp deletion in exon 2.
Key facts
- Cell type
HEK-293T
- Species or organism
Human
- Tissue
Kidney
- Form
Liquid
- Knockout validation
Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 2 and 5 bp deletion in exon 2
- Antibiotic resistance
G418 (Geneticin) 1µg/mL
- Concentration
Properties
- Gene name
CD47
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Immunocytochemistry, Sanger Sequencing, Western blot
Quality control
- STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
EU: 2 US: 2
- 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
Recommended control: Human wild-type HEK293T cell line (Human wild-type HEK-293T cell line ab255449). 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.
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
CD47 also referred to as integrin-associated protein carries a molecular weight of approximately 50 kDa. This protein is broadly expressed across various cell types notably on erythrocytes leukocytes and endothelial cells. Its transmembrane glycoprotein structure allows it to perform various cellular functions. CD47 interacts with specific ligands prominently SIRPα which are present on immune cells such as macrophages and dendritic cells. The engagement of CD47 with these ligands plays a major role in cellular signaling processes.
- Biological function summary
CD47 functions as a "don't eat me" signal inhibiting phagocytosis by binding to SIRPα on macrophages. This protein is also important for cell adhesion and migration and can modulate interactions with integrins. While not a part of a large complex CD47 associates with various cytoskeletal and membrane proteins to maintain cellular architecture and transmission of mechanical forces. Additionally its interaction with thrombospondins influences angiogenesis and nitric oxide signaling.
- Pathways
CD47 is extensively involved in the regulation of the immune response and angiogenesis. The interaction between CD47 and SIRPα plays a part in the regulation of macrophage activity impacting the immune signaling pathway. CD47 also interacts with integrins allowing it to contribute to the angiogenesis pathway which is critical in the formation of new blood vessels. These interactions help coordinate complex cellular responses and maintain tissue homeostasis.
- Associated diseases and disorders
CD47 levels have associations with cancer and chronic inflammatory diseases. In cancer CD47 overexpression can allow tumor cells to evade the immune response by downregulating phagocytosis through SIRPα interaction. Therapeutically targeting CD47 using agents like anti-CD47 antibodies shows potential for enhancing anti-tumor immunity. Additionally CD47 is involved in atherosclerosis where its interaction with thrombospondin-1 contributes to disease pathogenesis by affecting nitric oxide signaling and vascular remodeling.
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7 product images
Immunocytochemistry - Human CD47 knockout HEK-293T cell line (ab266324)
Anti-CD47 antibody [IAP/964] - BSA and Azide free ab213079 staining CD47 in wild-type HEK293 cells (top panel) and CD47 knockout HEK293 cells (bottom panel) (ab266324). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-CD47 antibody [IAP/964] - BSA and Azide free ab213079 at 0.4 μg/ml concentration and Anti-beta Tubulin antibody - Loading Control ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Western blot - Human CD47 knockout HEK-293T cell line (ab266324)
Lanes 1-4: Merged signal (red and green). Green - Anti-CD47 antibody [EPR21794] ab218810 observed at 47-52 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.
Anti-CD47 antibody [EPR21794] ab218810 Anti-CD47 antibody [EPR21794] was shown to specifically react with CD47 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266324 (knockout cell lysate Human CD47 knockout HEK-293T cell lysate ab257220) was used. Wild-type and CD47 knockout samples were subjected to SDS-PAGE. Anti-CD47 antibody [EPR21794] ab218810 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4° at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD47 antibody [EPR21794] (Anti-CD47 antibody [EPR21794] ab218810) at 1/500 dilution
Lane 1: Wild-type HEK293T cell lysate at 20 µg
Lane 2: CD47 knockout HEK293T cell lysate at 20 µg
Lane 3: Jurkat cell lysate at 20 µg
Lane 4: HepG2 cell lysate at 20 µg
SecondaryAll lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution
Predicted band size: 35 kDa
Observed band size: 47-52 kDa
This data was developed using the same antibody clone in a different buffer formulation (Anti-CD47 antibody [EPR21794] ab218810).
Lanes 1-4: Merged signal (red and green). Green - Anti-CD47 antibody [EPR21794] ab218810 observed at 47-52 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.
Anti-CD47 antibody [EPR21794] ab218810 Anti-CD47 antibody [EPR21794] was shown to specifically react with CD47 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266324 (knockout cell lysate Human CD47 knockout HEK-293T cell lysate ab257220) was used. Wild-type and CD47 knockout samples were subjected to SDS-PAGE. Anti-CD47 antibody [EPR21794] ab218810 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Cell Culture - Human CD47 knockout HEK-293T cell line (ab266324)
Representative images of CD47 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.
Immunocytochemistry/ Immunofluorescence - Human CD47 knockout HEK-293T cell line (ab266324)
Immunocytochemistry analysis of CD47 KO HEK293T (ab266324) cells labelling CD47 with Anti-CD47 antibody [EPR23002-67] ab256495 at 1:100 (5.62 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% TritonX-100. Cells were counterstained with Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889) at 1/200 (2.5μg/ml) dilution. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used as the secondary antibody at 1:1000 (2 ug/ml) dilution. DAPI (blue) was used as nuclear counterstain. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used as the secondary antibody only control.
Confocal image showing membranous staining in Parental HEK293 cell line, no staining in CD47 KO HEK293T cell line.
Sanger Sequencing - Human CD47 knockout HEK-293T cell line (ab266324)
Allele-2: 5 bp deletion in exon 2.
Sanger Sequencing - Human CD47 knockout HEK-293T cell line (ab266324)
Allele-1: 11 bp deletion in exon 2
Sanger Sequencing - Human CD47 knockout HEK-293T cell line (ab266324)
Sequencing chromatogram displaying sequence edit in exon 2
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