Human CXCL10 (IP10) knockout A549 cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human CXCL10 (IP10) knockout A549 cell line (AB266971)
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° 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.
All lanes:
Western blot - Anti-IP10 antibody [EPR20764] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr20764-ab214668'>ab214668</a>) at 1/1000 dilution
Lane 1:
Wild-type A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg
Lane 2:
Wild-type A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100 ng/ml, 32 h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10 ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg
Lane 2:
Western blot - Human CXCL10 (IP10) knockout A549 cell line (ab266971)
Lane 3:
IP10 knockout A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg
Lane 4:
IP10 knockout A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100ng/ml, 32h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg
Lane 5:
THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg
Lane 6:
THP-1 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg
Predicted band size: 10 kDa
Observed band size: 11 kDa
false
- WB
Lab
Western blot - Human CXCL10 (IP10) knockout A549 cell line (AB266971)
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° 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.
All lanes:
Western blot - Anti-IP10 antibody [EPR24674-12] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr24674-12-ab283681'>ab283681</a>) 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 (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 32 hours and 10 ng/m TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) for 32 hours, and 5ug/ml Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) for the last 6 hours, whole cell lysate at 40 µg
Lane 2:
Western blot - Human CXCL10 (IP10) knockout A549 cell line (ab266971)
Lane 3:
Untreated IP10 knockout A549 whole cell lysate at 40 µg
Lane 4:
IP10 knockout A549 treated with 100 ng/ml IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 32 hours and 10 ng/m TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) for 32 hours, and 5ug/ml Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) 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 (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution
Predicted band size: 10 kDa
Observed band size: 11 kDa
false
- ICC/IF
Supplier Data
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.
- Cell Culture
Lab
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.
- Sanger seq
Unknown
Sanger Sequencing - Human CXCL10 (IP10) knockout A549 cell line (AB266971)
Allele-2 : 1 bp insertion in exon 2.
- Sanger seq
Unknown
Sanger Sequencing - Human CXCL10 (IP10) knockout A549 cell line (AB266971)
Allele-1 : 4 bp deletion in exon2
Complementary Products
Primary Antibodies
AB306587
Anti-CXCL10/IP-10 antibody [EPR24674-84]
BOND RX™ Validated|20ul selling size|RabMAb|Recombinant|KO Validated
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL10/IP-10 antibody [EPR24674-84] (AB306587)](https://content.abcam.com/products/images/cxcl10-ip-10-antibody-epr24674-84-ab306587--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img403870.jpg)
- 0
- 0
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, 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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
Culture medium
F-12K + 10% FBS
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
IP10 plays a role in modulating the activities of immune cells. It attracts T cells eosinophils monocytes and natural killer (NK) cells by binding to the CXCR3 receptor. IP10 is not part of a larger complex but interacts with other cytokines to influence cell migration and the immune response. High levels of IP10 can reflect strong immune activation which is why it is often measured in inflammatory conditions using standard assays like the IP-10 ELISA kits.
Pathways
The role of IP10 lies within the Th1-type immune response pathway. In this pathway IP10 works alongside other chemokines to recruit and activate immune cells to sites of inflammation or infection. It synergizes with IFN-γ to propagate immune signals. IP10 is also linked with the CXCR3 receptor which plays a critical role in these pathways providing a connection to other proteins such as CXCL9 and CXCL11 which have similar functions in cell-mediated immunity.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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