Product datasheet

Human ABCC1 (MRP1) knockout HeLa cell line ab265256
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製品の概要
製品名

Human ABCC1 (MRP1) knockout HeLa cell line

Parental Cell LineHeLaOrganism

HumanMutation description

Knockout achieved by using CRISPR/Cas9, 14 bp deletion in exon 9 and 2 bp deletion in exon 9

Passage number<20

Knockout validation

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

アプリケーション

適用あり : WB, Flow Cyt, ICC

Biosafety level

2

特記事項

Recommended control: Human wild-type HeLa cell line (ab255928). 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 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 ahaemocytometer 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, ifrequired.
1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

Adherent

TissueCervixCell typeepithelial

DiseaseAdenocarcinoma

GenderFemale

STR Analysis

Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18
TH01: 7 TPOX: 8,12 CSF1PO: 9, 10

Antibiotic resistancePuromycin 1.00µg/ml

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

Mediates export of organic anions and drugs from the cytoplasm. Mediates ATP-dependenttransport of glutathione and glutathione conjugates, leukotriene C4, estradiol-17-beta-oglucuronide, methotrexate, antiviral drugs and other xenobiotics. Confers resistance to anticancerdrugs. Hydrolyzes ATP with low efficiency.

組織特異性

Lung, testis and peripheral blood mononuclear cells.

配列類似性

Belongs to the ABC transporter superfamily. ABCC family. Conjugate transporter (TC 3.A.1.208)
subfamily.
Contains 2 ABC transmembrane type-1 domains.
Contains 2 ABC transporter domains.

細胞内局在

Cell membrane.

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ab265256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

WB

Abreviews

特記事項

Use at an assay dependent concentration. Predicted molecularweight: 171 kDa.
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アプリケーション

特記事項

Abreviews

Flow Cyt

Use at an assay dependent concentration.

ICC

Use at an assay dependent concentration.

画像

All lanes : Anti-MRP1 antibody [EPR21062] (ab233383) at 1/1000
dilution

Lane 1 : Wild-type HeLa (Human epithelial cell line from cervixadenocarcinoma) whole cell lysate

Lane 2 : MRP1 knockout HeLa (Human epithelial cell line fromcervix adenocarcinoma) whole cell lysateLane 3 : Wild-type A549 (Human lung carcinoma cell line) wholecell lysate

Lane 4 : MRP1 knockout A549 (Human lung carcinoma cell line)
whole cell lysate
Western blot - Human ABCC1 (MRP1) knockout
HeLa cell line (ab265256)

Lysates/proteins at 20 µg per lane.

SecondaryAll lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW)
preadsorbed (ab216773) at 1/20000 dilution
Performed under reducing conditions.

Predicted band size: 171 kDa
Observed band size: 250 kDa

Lanes 1-4: Merged signal (red and green). Green - ab233383
observed at 250 kDa. Red - loading control ab7291 observed at 50kDa.

ab233383 Anti-MRP1 antibody [EPR21062] was shown tospecifically react with MRP1 in wild-type HeLa cells. Loss of signal
was observed when knockout cell line ab265256 (knockout celllysate ab257242) was used. Wild-type and MRP1 knockout
samples were subjected to SDS-PAGE. ab233383 and Anti-alphaTubulin antibody [DM1A] - Loading Control (ab7291) wereincubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000
3dilution respectively. Blots were developed with Goat anti-Rabbit
IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat antiMouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
secondary antibodies at 1 in 20000 dilution for 1 hour at room
temperature before imaging.

ab233383 staining MRP1 in wild-type HeLa cells (top panel) andABCC1 knockout HeLa cells (ab265256) (bottom panel). 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 ab233383 at 1/100 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 ABCC1 (MRP1) knockout HeLa cell line

blue with DAPI.

(ab265256)

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

Flow cytometry overlay histogram showing wild-type HeLa (greenline) and ABCC1 knockout HeLa cells (ab265256) stained withab260038 (red line). The cells were fixed with 80% methanol (5min) and then permeabilized with 0.1% PBS-Triton X-100 for 15min. The cells were then incubated in 1x PBS containing 10%
normal goat serum to block non-specific protein-protein interactionfollowed by the antibody (ab260038) (1x106 in 100μl at 0.2 μg/ml)
for 30 min at 22°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor®
488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at22°C.
Flow Cytometry (Intracellular) - Human ABCC1(MRP1) knockout HeLa cell line (ab265256)

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730)
used at the same concentration and conditions as the primaryantibody (wild-type HeLa - black line ABCC1 knockout HeLa - greyline). Unlabelled sample was also used as a control (this line is notshown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Bluelaser (488nm) and 525/40 bandpass filter.

4Allele-1: 14 bp deletion in exon 9.

Sanger Sequencing - Human ABCC1 knockout HeLa
cell line (ab265256)

Allele-2: 2 bp deletion in exon 9.

Sanger Sequencing - Human ABCC1 knockout HeLa
cell line (ab265256)

Representative images of ABCC1 knockout HeLa cells, low andhigh confluency examples (top left and right respectively) and wildtype HeLa cells, low and high confluency (bottom left and rightrespectively) showing typical adherent, epithelial-like morphology.
Images were captured at 10X magnification using a EVOS XLCore microscope.

Cell Culture - Human ABCC1 (MRP1) knockout
HeLa cell line (ab265256)

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