Human ANPEP (CD13) knockout THP-1 cell line
- Advanced Validation
- What is this?
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(1 Publication)
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
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 ab7417 at 2.5μg/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).
This image was generated from the hybridoma version of the product.
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
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 ab108382 at 1/1000 dilution and 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) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (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).
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
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 ab108310 at 1/500 dilution and 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) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (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).
- Flow Cyt
Lab
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 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 (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) (ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG2aκ (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.
- Flow Cyt
Lab
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 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 (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) (ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG1κ (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.
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
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 ab227663 at 1μg/ml concentration and 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) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (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).
- WB
Lab
Western blot - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
Lanes 1 - 4 : Merged signal (red and green). Green - ab108310 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
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 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 ab108310 and 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-CD13 antibody [EPR4058] (<a href='/en-us/products/primary-antibodies/cd13-antibody-epr4058-ab108310'>ab108310</a>) 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
Predicted band size: 109 kDa
Observed band size: 160 kDa
false
- WB
Lab
Western blot - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
Lanes 1 - 4 : Merged signal (red and green). Green - ab108382 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
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 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 ab108382 and 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-CD13 antibody [EPR4059] (<a href='/en-us/products/primary-antibodies/cd13-antibody-epr4059-ab108382'>ab108382</a>) 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
Predicted band size: 109 kDa
Observed band size: 160 kDa
false
- WB
Lab
Western blot - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
Lanes 1 - 4 : Merged signal (red and green). Green - ab227663 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab227663 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 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 ab227663 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 400 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-CD13 antibody [SP187] (<a href='/en-us/products/primary-antibodies/cd13-antibody-sp187-ab227663'>ab227663</a>) at 1/400 dilution
Lane 1:
Wild-type THP-1 cell lysate at 30 µg
Lane 2:
Western blot - Human ANPEP (CD13) knockout THP-1 cell lysate (<a href='/en-us/products/cell-lysates/human-anpep-cd13-knockout-thp-1-cell-lysate-ab275505'>ab275505</a>) at 30 µg
Lane 3:
PANC-1 cell lysate at 30 µg
Lane 4:
HEK-293 cell lysate at 30 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
Predicted band size: 109 kDa
Observed band size: 160 kDa,37 kDa
false
- Sanger seq
Supplier Data
Sanger Sequencing - Human ANPEP (CD13) knockout THP-1 cell line (AB273759)
Homozygous : 2 bp deletion in exon 2
Complementary Products
Reactivity data
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
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.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
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.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Suspension
Gender
Male
Target data
Publications (1)
Recent publications for all applications. Explore the full list and refine your search
iScience 26:108219 PubMed37942010
2023
Applications
Unspecified application
Species
Unspecified reactive species
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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