Human CXCL10 (IP10) knockout A549 cell line (ab266971)
Overview
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Product name
Human CXCL10 (IP10) knockout A549 cell line
See all IP10 lysates -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and 4 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: ICC/IF, WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A549 cell line (ab255450). 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: F-12K + 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 2x103-1x104 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 6x104 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.
- Do not exceed 7x104 cells/cm2.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
STR Analysis
Amelogenin X,Y D5S818: 11 D13S317: 11 D7S820: 8, 11 D16S539: 11, 12 vWA: 14 TH01: 8,9.3 TPOX: 8,11 CSF1PO: 10, 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
Target
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Function
Chemotactic for monocytes and T-lymphocytes. Binds to CXCR3. -
Sequence similarities
Belongs to the intercrine alpha (chemokine CxC) family. -
Post-translational
modificationsCXCL10(1-73) is produced by proteolytic cleavage after secretion from keratinocytes. -
Cellular localization
Secreted. - Information by UniProt
Associated products
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Biochemicals
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KO cell lysates
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab266971 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/IF |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration. Predicted molecular weight: 10 kDa.
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| Notes |
|---|
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ICC/IF
Use at an assay dependent concentration. |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 10 kDa. |
Images
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Western blot - Human CXCL10 (IP10) knockout A549 cell line (ab266971)All lanes : Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution
Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 2 : Wild-type A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 3 : IP10 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 4 : IP10 knockout A549 IFN-y (ab259377) (100ng/ml, 32h) and TNF-alpha (ab259410) (10ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 5 : THP-1 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 6 : THP-1 IFN-y (ab259377) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Predicted band size: 10 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?Lanes 1 - 6: Merged signal (red and green). Green - ab214668 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab214668 was shown to react with IP10 in wild-type A549 cells in western blot with loss of signal observed in IP10 knockout cell line ab266971 (knockout cell lysate ab256888). Wild-type and IP10 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214668 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C 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® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Western blot - Human CXCL10 (IP10) knockout A549 cell line (ab266971)All lanes : Anti-IP10 antibody [EPR24674-12] (ab283681) at 1/1000 dilution
Lane 1 : Untreated Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate at 40 µg
Lane 2 : Wild-type A549 treated with 100 ng/ml IFN-y (ab259377) for 32 hours and 10 ng/m TNF-alpha (ab259410) for 32 hours, and 5ug/ml Brefeldin A (ab120299) for the last 6 hours, whole cell lysate at 40 µg
Lane 3 : Untreated IP10 knockout A549 whole cell lysate at 40 µg
Lane 4 : IP10 knockout A549 treated with 100 ng/ml IFN-y (ab259377) for 32 hours and 10 ng/m TNF-alpha (ab259410) for 32 hours, and 5ug/ml Brefeldin A (ab120299) for the last 6 hours, whole cell lysate at 40 µg
Lane 5 : Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg
Lane 6 : THP-1 treated with 200ng/ml IFN-y (ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours, whole cell lysate at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution
Predicted band size: 10 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST
Lanes 1-6: Merged signal (red and green). Green - ab283681 observed at 11 kDa. Red-loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) was observed at 36 kDa.
ab283681 Anti-TNF Receptor I antibody [EPR24674-12] was shown to specifically react with IP10 in treated wild-type A549 cells. Loss of signal was observed when IP10 knockout cell lines ab266971 (knockout cell lysate ab256888) were used. Wild-type and IP10 knockout samples were subjected to SDS-PAGE. ab283681 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 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 10000 dilution for 1 hour at room temperature before imaging.
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Immunocytochemistry/ Immunofluorescence - Human CXCL10 (IP10) knockout A549 cell line (ab266971)Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CXCL10 KO A549 (ab266971) cells labelling IP10 with ab283681 at 1/50 (12.86 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing the signal expression was increased in Parental A549 cells after treatment with Interferon gamma (200 ng/ml) and lipopolysaccharide (50 ng/ml) for 3h, then adding Brefeldin A (1 ug/ml) for another 21h, and no staining in treated CXCL10 KO A549 cells with the same conditions. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.
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Sanger Sequencing - Human CXCL10 knockout A549 cell line (ab266971)Allele-1: 4 bp deletion in exon2
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Sanger Sequencing - Human CXCL10 knockout A549 cell line (ab266971)Allele-2: 1 bp insertion in exon 2.
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Cell Culture - Human CXCL10 (IP10) knockout A549 cell line (ab266971)Representative images CXCL10 knockout A549 cells, low and high confluency examples (top left and right respectively) and wild-type A549 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 M5000 microscope.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266971 has not yet been referenced specifically in any publications.