Human ANPEP (CD13) knockout THP-1 cell line (ab273759)

ANPEP 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, Homozygous: 2 bp deletion in exon 2.

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Images

Flow Cytometry - Human ANPEP (CD13) knockout THP-1 cell line (AB273759), expandable thumbnail

Publications

Key facts

Cell type: THP-1
Species or organism: Human
Tissue: Blood
Form: Liquid
Knockout validation: Immunocytochemistry, Sanger Sequencing, Western blot
Mutation description: Knockout achieved by using CRISPR/Cas9, Homozygous: 2 bp deletion in exon 2

Alternative names

AMPN_HUMAN, ANPEP, AP-M, AP-N, Alanyl (membrane) aminopeptidase, Alanyl aminopeptidase, Aminopeptidase M, Aminopeptidase N, CD13 antigen, LAP 1, Microsomal aminopeptidase, Myeloid plasma membrane glycoprotein CD13, P150, PEPN, gp150, hAPN

Key facts

Cell type: THP-1
Species or organism: Human
Tissue: Blood
Form: Liquid
Knockout validation: Immunocytochemistry, Sanger Sequencing, Western blot
Mutation description: Knockout achieved by using CRISPR/Cas9, Homozygous: 2 bp deletion in exon 2
Disease: Acute Monocytic Leukemia
Concentration

Properties

Gene name: ANPEP
Gene editing type: Knockout
Gene editing method: CRISPR technology
Knockout validation: Immunocytochemistry, 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: Suspension
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. Based on cell count, seed cells in an appropriate cell culture flask at a density of 4x10⁵ cells/mL. 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.
5. THP-1 cells recover slowly from cryopreservation and therefore may not be ready for subculture for a number of days. Cells should be left as much as possible over this time and only subcultured when the cell density reaches 8x10⁵ cells/mL.Small amounts of fresh media can be added until cell number/viability improves.

Subculture guidelines

All seeding densities should be based on cell counts gained by established methods.
Cells should be seeded at 2x10⁵ - 3x10⁵ cells/mL and subcultured when they have reached 8x10⁵ cells/mL.
It is not recommended to allow the cell density to exceed 1x10⁶ cells/mL.

Culture medium

RPMI + 10% FBS + 0.05 mM beta-mercaptoethanol

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 THP-1 cell line (ab275477). 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.

Upon thawing make banks as soon as possible and use each bank within 10-20 passages

As cell line growth can vary please attempt culture for 2 weeks from revival of initial bank before contacting the technical team.

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

CD13 also known as aminopeptidase N (ANPEP) is a transmembrane protein with a molecular weight of approximately 150 kDa. It functions as a zinc-dependent metalloenzyme which cleaves N-terminal amino acids from peptides and proteins. CD13 protein is expressed on various cell types including myeloid cells epithelial cells endothelial cells and fibroblasts. Its presence is significantly observed in the brush border of the small intestinal mucosa and the renal proximal tubule. Researchers can study CD13 using anti-CD13 antibodies and CD13 ELISA kits.

Biological function summary

CD13 regulates peptide-mediated signaling and controls the maturation and catabolism of bioactive peptides. The enzymatic function of CD13 influences processes such as cell proliferation motility and angiogenesis. It does not operate as part of a larger complex but its activity modulates several cellular and systemic functions. CD13's role in these biological processes highlights its importance in modulating local and systemic peptide pools which contributes to its diverse physiological effects.

Pathways

CD13 plays significant roles in the renin-angiotensin system and the regulation of inflammatory responses. In the renin-angiotensin system CD13 modulates the activity of angiotensin influencing blood pressure and fluid balance. Through its enzymatic activity CD13 interacts with proteins such as ACE another critical player in this pathway. In inflammation CD13 regulates the availability of chemotactic peptides affecting leukocyte migration and adhesion. The proteins it works with in inflammatory pathways include cytokines which CD13 indirectly modulates by altering chemokine activity.

Associated diseases and disorders

Aberrant CD13 expression and activity are implicated in cancer and inflammatory diseases. In cancer overexpression of CD13 correlates with tumor growth and metastasis particularly in cancers like renal cell carcinoma and prostate cancer. It can confer an increased malignant phenotype by promoting angiogenesis and immune evasion. In inflammatory disorders dysregulation of CD13 exacerbates diseases such as rheumatoid arthritis through its impact on peptide-mediated signaling and immune cell movement. Its connection with inflammatory cytokines like IL-8 indicates its important role in mediating inflammatory responses.

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9 product images

Flow Cytometry - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Flow cytometry overlay histogram showing wild-type THP1 (green line) and ANPEP knockout THP1 cells (ab273759) stained with Anti-CD13 antibody [WM15] ab7417 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (Anti-CD13 antibody [WM15] ab7417) (1x10⁶ in 100μl at 1 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG1κ (Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control ab170190) used at the same concentration and conditions as the primary antibody (wild-type THP1 cells - black line; ANPEP knockout THP1 cells ab273759 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
This image was generated from the hybridoma version of the product.
Flow Cytometry - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Flow cytometry overlay histogram showing wild-type THP1 (green line) and ANPEP knockout THP1 cells (ab273759) stained with Anti-CD13 antibody [22A5] ab20136 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (Anti-CD13 antibody [22A5] ab20136) (1x10⁶ in 100μl at 1 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG2aκ (Mouse IgG2a, Kappa Monoclonal [MOPC-173] -Isotype Control - ChIP Grade ab18413) used at the same concentration and conditions as the primary antibody (wild-type THP1 cells - black line; ANPEP knockout THP1 cells ab273759 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Anti-CD13 antibody [EPR4058] ab108310 staining CD13 in wild-type THP-1 cells (top panel) and ANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 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-CD13 antibody [EPR4058] ab108310 at 1/500 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha 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 rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) 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).
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Anti-CD13 antibody [SP187] ab227663 staining CD13 in wild-type THP-1 cells (top panel) and ANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 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-CD13 antibody [SP187] ab227663 at 1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha 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 rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) 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).
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Anti-CD13 antibody [EPR4059] ab108382 staining CD13 in wild-type THP-1 cells (top panel) and ANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 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-CD13 antibody [EPR4059] ab108382 at 1/1000 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha 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 rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) 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 ANPEP (CD13) knockout THP-1 cell line (ab273759)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD13 antibody [EPR4059] ab108382 observed at 160 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
Anti-CD13 antibody [EPR4059] ab108382 was shown to react with CD13 in wild-type THP-1 cells in western blot with loss of signal observed in ANPEP knockout cell line ab273759 (knockout cell lysate Human ANPEP (CD13) knockout THP-1 cell lysate ab275505). Wild-type and ANPEP knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with Anti-CD13 antibody [EPR4059] ab108382 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4° at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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-CD13 antibody [EPR4059] (Anti-CD13 antibody [EPR4059] ab108382) at 1/1000 dilution
Lane 1: Wild-type THP-1 cell lysate at 30 µg
Lane 2: ANPEP knockout THP-1 cell lysate at 30 µg
Lane 2: Western blot - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Lane 3: PANC-1 cell lysate at 30 µg
Lane 4: HEK-293 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 109 kDa
Observed band size: 160 kDa
Western blot - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD13 antibody [EPR4058] ab108310 observed at 160 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
Anti-CD13 antibody [EPR4058] ab108310 was shown to react with CD13 in wild-type THP-1 cells in western blot with loss of signal observed in ANPEP knockout cell line ab273759 (knockout cell lysate Human ANPEP (CD13) knockout THP-1 cell lysate ab275505). Wild-type and ANPEP knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with Anti-CD13 antibody [EPR4058] ab108310 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4° at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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-CD13 antibody [EPR4058] (Anti-CD13 antibody [EPR4058] ab108310) at 1/1000 dilution
Lane 1: Wild-type THP-1 cell lysate at 30 µg
Lane 2: ANPEP knockout THP-1 cell lysate at 30 µg
Lane 2: Western blot - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Lane 3: PANC-1 cell lysate at 30 µg
Lane 4: HEK-293 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 109 kDa
Observed band size: 160 kDa
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Anti-CD13 antibody [WM15] ab7417 staining CD13 in wild-type THP-1 cells (top panel) and ANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 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-CD13 antibody [WM15] ab7417 at 2.5μg/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).
This image was generated from the hybridoma version of the product.
Sanger Sequencing - Human ANPEP (CD13) knockout THP-1 cell line (ab273759)
Homozygous: 2 bp deletion in exon 2