Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (ab273379)
Overview
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Product name
Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line -
Parental Cell Line
A431 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 49 bp deletion in exon 5. -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WB, Sandwich ELISA, ICC/IFmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A-431 cell line (ab275462).
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: McCoY5a + 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 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, if required.
- 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 and relevant 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
Skin -
Cell type
epithelial -
Disease
Epidermoid Carcinoma -
Gender
Female -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
Target
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Relevance
Interleukin 1 receptor antagonist inhibits the activities of interleukin 1 alpha and beta, and modulates a variety of interleukin 1 related immune and inflammatory responses. This gene and five other related cytokine genes form a cluster spanning approximately 400 kb on chromosome 2. A polymorphism of this gene is associated with increased risk of developing osteoporosis and gastric cancer. Four alternatively spliced transcript variants encoding differentt isoforms have been reported.
Associated products
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ELISA kits
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab273379 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration.
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| Sandwich ELISA |
Use at an assay dependent concentration.
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| ICC/IF |
Use at an assay dependent concentration.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. |
|
Sandwich ELISA
Use at an assay dependent concentration. |
|
ICC/IF
Use at an assay dependent concentration. |
Images
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Sanger Sequencing - Human IL1RN knockout A-431 cell line (ab273379)
49 bp deletion in exon 5
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Western blot - Human IL1RN knockout A-431 cell line (ab273379)All lanes : Anti-IL-1RA antibody [EPR6483] (ab124962) at 1/10000 dilution
Lane 1 : Wild-type A431 cell lysate
Lane 2 : IL-1RA knockout A431 cell lysate
Performed under reducing conditions.
Observed band size: 19 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-IL-1RA antibody [EPR6483] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab124962 was shown to bind specifically to IL-1RA. A band was observed at 19 kDa in wild-type A431 cell lysates with no signal observed at this size in IL1RN knockout cell line ab273379 (knockout cell lysate ab275530). To generate this image, wild-type and IL1RN knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunocytochemistry - Human IL1RN knockout A-431 cell line (ab273379)IL-1RA staining observed in wild-type A431 cells (top panel) and IL1RN knockout A431 cells (bottom panel). 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 an anti-IL-1ra antibody at 10μg/ml concentration (shown in green) and ab195889 (Mouse monoclonal to alpha Tubulin - Alexa Fluor® 594) at 1/250 dilution (shown in red) overnight at 4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Western blot - Human IL1RN knockout A-431 cell line (ab273379)All lanes : Anti-IL-1ra antibody at 0.5 µg/ml
Lane 1 : Wild-type A431 cell lysate
Lane 2 : IL-1RA knockout A431 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : SK-OV-3 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 18 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - Anti-IL-1ra antibody observed at 18 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
Anti-IL-1ra antibody was shown to react with IL-1ra in wild-type A431 cells in Western blot with loss of signal observed in IL-1RA knockout cell line ab273379 (IL-1RA knockout cell lysate ab275530). Wild-type A431 and IL-1RA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with anti-IL-1ra antibody and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 0.5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Donkey anti-Goat IgG H&L (IRDye® 800CW) preabsorbed (ab216775) and Donkey anti-Mouse 680RD secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
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Sandwich ELISA - Human IL1RN knockout A-431 cell line (ab273379)Human IL-1RA concentration was interpolated from the IL-1RA standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human IL-1ra ELISA Kit (ab211650). Wild-type A-431, IL1RN knockout A-431 (ab273379) and THP-1 cells were assessed in duplicate (n=2). Data are represented as the mean and error bars represent standard deviation.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab273379 has not yet been referenced specifically in any publications.