Product datasheet

Human ANPEP (CD13) knockout THP-1 cell line ab273759
1 References 画像数 10

製品の概要
製品名

Human ANPEP (CD13) knockout THP-1 cell line

Parental Cell LineTHP-1Organism

HumanMutation description

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

Passage number<20

Knockout validation

Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB)

アプリケーション

適用あり : Flow Cyt, WB, ICC

Biosafety level

1

特記事項

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.

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

Culture medium: RPMI + 10% FBS + 0.05 mM β-mercaptoethanol
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phaseand 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 5minutes 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 2-4x105 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.

Subculture guidelines:
All seeding densities should be based on cell counts gained by established methods.
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Cells should be seeded at 2-4x105 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.
A partial media change 24 hours prior to subculture may be helpful to encourage growth, ifrequired.
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 andrelevant patents please refer to our limited use license and patent pages.
We will provide viable cells that proliferate on revival.

製品の特性

Number of cells

1 x 106 cells/vial, 1 mL

Adherent /Suspension

Suspension

TissueBlood

Cell type

acute monocytic leukemia

DiseaseAcute Monocytic Leukemia

GenderMale

Mycoplasma freeYes

保存方法

Shipped on Dry Ice. Store in liquid nitrogen.

バッファー

Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether

ターゲット情報機能Broad specificity aminopeptidase. Plays a role in the final digestion of peptides generated fromhydrolysis of proteins by gastric and pancreatic proteases. May play a critical role in thepathogenesis of cholesterol gallstone disease. May be involved in the metabolism of regulatory
peptides of diverse cell types including small intestinal and tubular epithelial cells, macrophages,
granulocytes and synaptic membranes from the CNS. Found to cleave antigen peptides bound tomajor histocompatibility complex class II molecules of presenting cells and to degradeneurotransmitters at synaptic junctions. Is also implicated as a regulator of IL-8 bioavailability inthe endometrium, and therefore may contribute to the regulation of angiogenesis. Is used as amarker for acute myeloid leukemia and plays a role in tumor invasion. In case of humancoronavirus 229E (HCoV-229E) infection, serves as receptor for HCoV-229E spike glycoprotein.
Mediates as well human cytomegalovirus (HCMV) infection.

組織特異性

Expressed in epithelial cells of the kidney, intestine, and respiratory tract; granulocytes,monocytes, fibroblasts, endothelial cells, cerebral pericytes at the blood-brain barrier, synaptic
membranes of cells in the CNS. Also expressed in endometrial stromal cells, but not in theendometrial glandular cells. Found in the vasculature of tissues that undergo angiogenesis and in
malignant gliomas and lymph node metastases from multiple tumor types but not in blood vesselsof normal tissues. A soluble form has been found in plasma. It is found to be elevated in plasmaand effusions of cancer patients.

配列類似性

Belongs to the peptidase M1 family.

ドメイン

Amino acids 260-353 are essential to mediate susceptibility to infection with HCoV-229E (inporcine/human chimeric studies) and more specifically amino acids 288-295 (mutagenesis2studies).
翻訳後修飾

Sulfated.
N- and O-glycosylated.
May undergo proteolysis and give rise to a soluble form.

細胞内局在

Cell membrane. Cytoplasm > cytosol. A soluble form has also been detected.

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab273759の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション

特記事項

AbreviewsFlow Cyt

Use at an assay dependent concentration.

WB

Use at an assay dependent concentration.

ICC

Use at an assay dependent concentration.

画像

Flow cytometry overlay histogram showing wild-type THP1 (greenline) and ANPEP knockout THP1 cells (ab273759) stained withab20136 (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 bythe antibody (ab20136) (1x106 in 100μl at 1 μg/ml) for 30 min at4°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor®
488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at4°C.
Isotype control antibody was mouse IgG2aκ (ab18413) used at theFlow Cytometry - Human ANPEP (CD13) knockout
THP-1 cell line (ab273759)

same concentration and conditions as the primary antibody (wildtype THP1 cells - black line; ANPEP knockout THP1 cellsab273759 - grey line). Unlabelled sample was also used as acontrol (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Bluelaser (488nm) and 525/40 bandpass filter.

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ab227663 staining CD13 in wild-type THP-1 cells (top panel) andANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells
were fixed with 4% paraformaldehyde (10 min) then permeabilizedwith 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 atroom temperature for 1h with a goat secondary antibody to rabbit
IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) anda goat secondary antibody to mouse IgG (Alexa Fluor® 594)
Immunocytochemistry/ Immunofluorescence -(ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in

Human ANPEP (CD13) knockout THP-1 cell line

blue with DAPI.

(ab273759)

Image was taken with a confocal microscope (Leica-Microsystems
TCS SP8).

All lanes : Anti-CD13 antibody [SP187] (ab227663) at 1/400
dilution

Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : ANPEP knockout THP-1 cell lysate
Lane 3 : PANC-1 cell lysate
Lane 4 : HEK-293 cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Western blot - Human ANPEP (CD13) knockout
THP-1 cell line (ab273759)

Observed band size: 160 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab227663
observed at 160 kDa. Red - loading control ab8245 (Mouse antiGAPDH 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 cellline ab273759 (knockout cell lysate ab275505). Wild-type andANPEP knockout THP-1 cell lysates were subjected to SDSPAGE. Membranes were blocked in 3% milk in TBS-T (0.1%4Tween®) before incubation with ab227663 and ab8245 (Mouseanti-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.

Flow cytometry overlay histogram showing wild-type THP1 (greenline) 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 FCreceptors 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 at4°C.
Isotype control antibody was mouse IgG1κ (ab170190) used at thesame concentration and conditions as the primary antibody (wildFlow Cytometry - Human ANPEP (CD13) knockout
THP-1 cell line (ab273759)

type THP1 cells - black line; ANPEP knockout THP1 cellsab273759 - grey line). Unlabelled sample was also used as acontrol (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Bluelaser (488nm) and 525/40 bandpass filter.

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 agoat secondary antibody to mouse IgG (Alexa Fluor® 594)
Immunocytochemistry/ Immunofluorescence -(ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in

Human ANPEP (CD13) knockout THP-1 cell line

blue with DAPI.

(ab273759)

Image was taken with a confocal microscope (Leica-Microsystems
TCS SP8).

5ab108310 staining CD13 in wild-type THP-1 cells (top panel) andANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells
were fixed with 4% paraformaldehyde (10 min) then permeabilizedwith 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 agoat secondary antibody to mouse IgG (Alexa Fluor® 594)
Immunocytochemistry/ Immunofluorescence -(ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in

Human ANPEP (CD13) knockout THP-1 cell line

blue with DAPI.

(ab273759)

Image was taken with a confocal microscope (Leica-Microsystems
TCS SP8).

ab7417 staining CD13 in wild-type THP-1 cells (top panel) andANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells
were fixed with 4% paraformaldehyde (10 min) then permeabilizedwith 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 atroom temperature for 1h with a goat secondary antibody to mouse
IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) anda goat secondary antibody to rabbit IgG (Alexa Fluor® 594)
Immunocytochemistry/ Immunofluorescence -(ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in

Human ANPEP (CD13) knockout THP-1 cell line

blue with DAPI.

(ab273759)

Image was taken with a confocal microscope (Leica-Microsystems
TCS SP8).

6

All lanes : Anti-CD13 antibody [EPR4059] (ab108382) at 1/1000
dilution

Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : ANPEP knockout THP-1 cell lysate
Lane 3 : PANC-1 cell lysate
Lane 4 : HEK-293 cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Western blot - Human ANPEP (CD13) knockout
THP-1 cell line (ab273759)

Observed band size: 160 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab108382
observed at 160 kDa. Red - loading control ab8245 (Mouse antiGAPDH 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 cellline ab273759 (knockout cell lysate ab275505). Wild-type andANPEP knockout THP-1 cell lysates were subjected to SDSPAGE. Membranes were blocked in 3% milk in TBS-T (0.1%Tween®) before incubation with ab108382 and ab8245 (Mouseanti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution
and a 1 in 20000 dilution respectively. Blots were incubated withGoat 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.

7All lanes : Anti-CD13 antibody [EPR4058] (ab108310) at 1/1000
dilution

Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : ANPEP knockout THP-1 cell lysate
Lane 3 : PANC-1 cell lysate
Lane 4 : HEK-293 cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Western blot - Human ANPEP (CD13) knockout
THP-1 cell line (ab273759)

Observed band size: 160 kDaLanes 1 - 4: Merged signal (red and green). Green - ab108310
observed at 160 kDa. Red - loading control ab8245 (Mouse antiGAPDH 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 cellline ab273759 (knockout cell lysate ab275505). Wild-type andANPEP knockout THP-1 cell lysates were subjected to SDSPAGE. Membranes were blocked in fluorescent western blot (TBSbased) blocking solution before incubation with ab108310 andab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots wereincubated 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.

Homozygous: 2 bp deletion in exon 2Sanger Sequencing - Human ANPEP (CD13)
knockout THP-1 cell line (ab273759)

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"

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