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  1. Link

    human-cd47-knockout-hek-293t-cell-line-ab266324.pdf

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Immunology Cell Type Markers CD Adhesion
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Human CD47 knockout HEK-293T cell line (ab266324)

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Sanger Sequencing - Human CD47 knockout HEK-293T cell line (ab266324)
  • Immunocytochemistry - Human CD47 knockout HEK-293T cell line (ab266324)
  • Western blot - Human CD47 knockout HEK293T cell line (ab266324)
  • Sanger Sequencing - Human CD47 knockout HEK293T cell line (ab266324)
  • Sanger Sequencing - Human CD47 knockout HEK293T cell line (ab266324)
  • Cell Culture - Human CD47 knockout HEK293T cell line (ab266324)

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Overview

  • Product name

    Human CD47 knockout HEK-293T cell line
  • Parental Cell Line

    HEK293T
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 2 and 5 bp deletion in exon 2
  • Passage number

    <20
  • Knockout validation

    Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB)
  • Tested applications

    Suitable for: ICC, WBmore details
  • Biosafety level

    2
  • General notes

    Recommended control: Human wild-type HEK293T 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.

    Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

    Culture medium: DMEM (High Glucose) + 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 2x104 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 2x104 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.

    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.

Properties

  • Number of cells

    1 x 106 cells/vial, 1 mL
  • Viability

    ~80%
  • Adherent /Suspension

    Adherent
  • Tissue

    Kidney
  • Cell type

    epithelial
  • STR Analysis

    Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12
  • Antibiotic resistance

    Puromycin 1.00µg/ml
  • Mycoplasma free

    Yes
  • Storage instructions

    Shipped on Dry Ice. Store in liquid nitrogen.
  • Storage buffer

    Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
  • Research areas

    • Immunology
    • Cell Type Markers
    • CD
    • Adhesion
    • Cancer
    • Tumor immunology
    • CD markers

Target

  • Function

    Has a role in both cell adhesion by acting as an adhesion receptor for THBS1 on platelets, and in the modulation of integrins. Plays an important role in memory formation and synaptic plasticity in the hippocampus (By similarity). Receptor for SIRPA, binding to which prevents maturation of immature dendritic cells and inhibits cytokine production by mature dendritic cells. Interaction with SIRPG mediates cell-cell adhesion, enhances superantigen-dependent T-cell-mediated proliferation and costimulates T-cell activation. May play a role in membrane transport and/or integrin dependent signal transduction. May prevent premature elimination of red blood cells. May be involved in membrane permeability changes induced following virus infection.
  • Tissue specificity

    Very broadly distributed on normal adult tissues, as well as ovarian tumors, being especially abundant in some epithelia and the brain.
  • Sequence similarities

    Contains 1 Ig-like V-type (immunoglobulin-like) domain.
  • Cellular localization

    Cell membrane.
  • Target information above from: UniProt accession Q08722 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt

Associated products

  • KO cell lysates

    • Human CD47 knockout HEK-293T cell lysate (ab257220)
  • KO cell pellets

    • Human CD47 knockout HEK-293T cell pellet (ab278890)
  • Related Products

    • Anti-CD47 antibody [EPR21794] (ab218810)
    • Anti-CD47 antibody [EPR21794] - BSA and Azide free (ab233122)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab266324 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Predicted molecular weight: 35 kDa.
Notes
ICC
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Predicted molecular weight: 35 kDa.

Images

  • Sanger Sequencing - Human CD47 knockout HEK-293T cell line (ab266324)
    Sanger Sequencing - Human CD47 knockout HEK-293T cell line (ab266324)
    Sequencing chromatogram displaying sequence edit in exon 2
  • Immunocytochemistry - Human CD47 knockout HEK-293T cell line (ab266324)
    Immunocytochemistry - Human CD47 knockout HEK-293T cell line (ab266324)

    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 ab213079 at 0.4 μg/ml concentration and 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) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (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 HEK293T cell line (ab266324)
    Western blot - Human CD47 knockout HEK293T cell line (ab266324)
    All lanes : Anti-CD47 antibody [EPR21794] (ab218810) at 1/500 dilution

    Lane 1 : Wild-type HEK293T cell lysate
    Lane 2 : CD47 knockout HEK293T cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : HepG2 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : 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 why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab218810 observed at 47-52 kDa. Red - loading control ab8245 observed at 36 kDa.

    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 ab257220) was used. Wild-type and CD47 knockout samples were subjected to SDS-PAGE. ab218810 and 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 (ab216773) and 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.

     

  • Sanger Sequencing - Human CD47 knockout HEK293T cell line (ab266324)
    Sanger Sequencing - Human CD47 knockout HEK293T cell line (ab266324)

    Allele-1: 11 bp deletion in exon 2

     

  • Sanger Sequencing - Human CD47 knockout HEK293T cell line (ab266324)
    Sanger Sequencing - Human CD47 knockout HEK293T cell line (ab266324)

    Allele-2: 5 bp deletion in exon 2.

     

  • Cell Culture - Human CD47 knockout HEK293T cell line (ab266324)
    Cell Culture - Human CD47 knockout HEK293T 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.

     

     

Protocols

  • Hemocytometer protocol
  • Mammalian cell tissue culture techniques protocol

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab266324? Please let us know so that we can cite the reference in this datasheet.

ab266324 has not yet been referenced specifically in any publications.

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